Methods of treating impaired glucose metabolism via administration of algal biomass

ABSTRACT

The invention is directed to methods of using  Chlorella protothecoides  microalgal biomass in the treatment of individuals having impaired glucose metabolism. In some cases, the patient has impaired fasting glucose, impaired glucose tolerance, or diabetes. In some methods, algal biomass is used to reduce blood glucose and/or body fat in a subject, or to increase the relative abundance of beneficial gut microflora in a subject. In preferred embodiments, the biomass is derived from  Chlorella protothecoides  cultures grown heterotrophically in which the algal cells comprise at least 15% algal oil by dry weight.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/254,035,filed Sep. 27, 2011, which is a US National Stage Application ofPCT/US2010/026117, filed Mar. 3, 2010, which claims the benefit under 35U.S.C. 119(e) of U.S. Provisional Patent Application No. 61/157,170,filed Mar. 3, 2009. Each of these applications is incorporated herein byreference in its entirety for all purposes.

REFERENCE TO A SEQUENCE LISTING

This application includes an electronic sequence listing in a file named“445930-Sequence.txt”, created on Apr. 30, 2014 and containing 2,994bytes, which is hereby incorporated by reference in its entirety for allpurposes.

FIELD OF THE INVENTION

The invention resides in the fields of medicine, aquaculture,fermentation, and genetic engineering.

BACKGROUND OF THE INVENTION

Impaired glucose tolerance is a pre-diabetic state of dysglycemia,associated with insulin resistance and increased risk of cardiovasculardisease and death. Barr et al., Circulation 116(2):151-157 (2007). Inmany cases, impaired glucose tolerance precedes the onset of type 2diabetes, a disease characterized by insulin resistance, relativeinsulin deficiency, and hyperglycemia. Id.

Hyperglycemia is consistently associated with cardiovascular disease.Mazzone et al., Lancet 371:1800-1809 (2008). High blood glucose levelscan lead to vascular complications by several mechanisms, resulting inatherosclerosis, among other complications. Id. As the incidence ofobesity and type 2 diabetes continue to rise throughout many parts ofthe world, effective methods for combating the deleterious effects ofhyperglycemia and preventing its occurrence are the focus of extensiveresearch.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a method of treatinga patient having impaired glucose metabolism by administering to thepatient an effective treatment regime of Chlorella protothecoidesbiomass comprising at least 15% oil by dry weight. In some cases, thebiomass contains at least 25%, 50% or 60% oil by dry weight. In somecases, the biomass contains 15-90%, 25-75%, 40-75% or 50-70% of algaloil by dry weight. In various embodiments, the patient, prior to beingplaced on the regimen, has been diagnosed with one or more conditionsselected from the group consisting of impaired glucose tolerance,dysglycemia, insulin resistance, cardiovascular disease, diabetes(including but not limited to types 1, 1.5, 2, and 3), metabolicsyndrome, hyperglycemia, and insulin deficiency.

In some embodiments, Chlorella protothecoides biomass used in themethods of the invention comprises oil in which at least 50% by weightof the oil is monounsaturated oil. In some cases, at least 50% by weightof the algal oil is oleic acid. In some cases, less than 5% by weight ofthe oil is docosahexanoic acid (DHA). In some embodiments, less than 1%by weight of the oil is DHA. In some cases, the oil is predominantlyencapsulated in cells of the Chlorella protothecoides biomass.

In some embodiments, the patient treated via the administration of aneffective regime of Chlorella protothecoides biomass is a patient havingimpaired glucose tolerance. In some cases, the patient has an impairedfasting glucose. In some embodiments, the patient has type 2 diabetes.In other cases, the patient has type 1 diabetes. In some cases, thepatient has type 1.5 (type 3) diabetes.

In some embodiments of the present invention, the patient receives analternative treatment, e.g., for impaired glucose tolerance,dysglycemia, insulin resistance, cardiovascular disease, diabetes(including but not limited to types 1, 1.5, 2, and 3), metabolicsyndrome, hyperglycemia, and/or insulin deficiency, before theadministering step and the alternative treatment is reduced oreliminated after the administering step. In some cases, the alternativetreatment is administration of a non-algal pharmaceutical product.

In some methods in accordance with the present invention, the Chlorellaprotothecoides biomass regime lowers the mean plasma glucoseconcentration of the patient relative to the concentration before theadministering step. In some cases, the mean plasma glucose concentrationis lowered 10-50%. In some embodiments, the methods of the presentinvention further comprise monitoring blood glucose levels of thepatient.

In some embodiments, the algal biomass regime lowers the percentage fatof total body weight in the patient relative to the percentage beforethe administering step.

In various embodiments, the algal biomass used in the methods of thepresent invention is derived from Chlorella protothecoides cultured anddried under good manufacturing practice (GMP) conditions. In some cases,the biomass is administered in the form of a tablet or capsule. In othercases, the biomass is administered in the form of a food product. Insome embodiments, the food product comprises at least 50% algal biomassby weight.

In one aspect, the present invention is directed to a method of reducingblood glucose in a subject by administering to the subject an effectiveregime of Chlorella protothecoides biomass comprising at least 15% oilby dry weight, whereby the mean blood glucose level of the subject isreduced relative to the level before administering the regime. In someembodiments, the method further comprises monitoring the blood glucoselevel of the subject prior to administering the regime to detect thecondition in need of treatment and then at various times after theregime is administered to detect the reduction. In various embodiments,the composition of the biomass or oil can be as described above, orthroughout the specification.

In one aspect, the present invention is directed to a method of reducingbody fat in a subject by administering to the subject an effectiveregime of Chlorella protothecoides biomass comprising at least 15% oilby dry weight, whereby the percentage fat of total body weight of thesubject is reduced relative to the percentage fat of total body weightbefore administering the regime. In some cases, the subject has a bodymass index of greater than 24.9. In some cases, the subject has a bodymass index of greater than 29.9. In some embodiments, the method furthercomprises monitoring the percentage fat of total body weight in thesubject prior to administering the regime to detect the condition inneed of treatment and then at various times after the regime isadministered to detect the reduction. In various embodiments, thecomposition of the biomass or oil can be as described above, orthroughout the specification.

In one aspect, the present invention is directed to a method ofincreasing the relative abundance of beneficial gut microflora in asubject by administering to the subject an effective regime of Chlorellaprotothecoides biomass comprising at least 15% oil by dry weight,whereby the relative abundance of beneficial gut microflora is increasedas compared to the relative abundance before administering the regime.In some cases, the beneficial gut microflora are of the classLactobacillales. In some embodiments, the method further comprisesmonitoring the relative abundance of gut microflora in the subject priorto administering the regime to detect the condition in need of treatmentand then at various times after the regime is administered to detect theincrease. In various embodiments, the composition of the biomass or oilcan be as described above, or throughout the specification.

In any one of the methods described above, the Chlorella protothecoidesbiomass regime can comprise administering the biomass at a dose of 1-20%of food by weight or calories. In some embodiments, the biomass isadministered daily for at least a week, a month, a year, or for life. Insome cases, the biomass is administered three times daily. In somecases, the algal biomass is administered proximate in time to intake ofa meal. In some embodiments, the algal biomass is administered with atleast one other edible ingredient as a food composition.

In any one of the methods described above, the biomass can be derivedfrom a culture of the microalgae Chlorella protothecoides, including anystrain of Chlorella protothecoides such as UTEX 1806, UTEX 411, UTEX264, UTEX 256, UTEX 255, UTEX 250, UTEX 249, UTEX 31, UTEX 29, UTEX 25,CCAP 211/17 and CCAP 211/8d. Chlorella protothecoides strains have the23S rRNA sequence of SEQ ID NOs: 5 or 6.

In any one of the methods described above, the microalgae can be grownheterotrophically. In some cases, microalgae are grown in a culturemedium containing depolymerized cellulosic material. In some cases, themicroalgae are grown in a culture medium containing a feedstockcomprising at least one carbon substrate selected from the groupconsisting of cellulosic material, a 5-carbon sugar, and a 6-carbonsugar. In various embodiments, the carbon substrate can be selected fromthe group consisting of glucose, xylose, sucrose, fructose, arabinose,mannose, galactose, and any combination thereof.

In any one of the methods described above in which the at least onecarbon substrate includes sucrose, the culture medium can optionallyfurther include at least one sucrose utilization enzyme. In some cases,the at least one sucrose utilization enzyme is an exogenous enzymeintroduced into the culture medium.

Some methods include first determining whether a patient or subject hasa condition, is in need of treatment, or would otherwise benefit fromadministration of the compositions discussed herein. For example, somemethods include first determining whether a patient or subject hasimpaired glucose metabolism, is in need of treatment to reduce body fat,or would benefit from a greater abundance of beneficial gut microflora.This determination can be made prior to administration of a regime orcomposition discussed herein.

Any two or more of the various embodiments described above or herein canbe combined together to produce additional embodiments encompassedwithin the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows dry cell weight per liter of multiple strains of Chlorellaprotothecoides when cultured in the presence of various types ofglycerol with additional glucose, wherein glycerol is added sequentiallyor in combination with glucose.

FIG. 2 shows a synergistic effect of a combination of xylose and glucoseon growth of Chlorella compared to xylose or glucose alone.

FIG. 3 shows growth of Chlorella protothecoides on glucose and fructose.

FIG. 4 shows plasma glucose, cholesterol and triglyceride concentrationsin Syrian golden hamsters that had consumed a hyperglycemia-inducingdiet with and without algal biomass from cultured Chlorellaprotothecoides. Values are mean±SEM and bars with differing lettersuperscripts are different from one another at P<0.05 (one-way ANOVA).

FIG. 5 shows plasma insulin concentrations in Syrian golden hamstersthat had consumed a hyperglycemia-inducing diet with and without algalbiomass from cultured Chlorella protothecoides. Values are mean±SEM anddiffering letters indicate difference at P<0.05 (one-way ANOVA).

FIG. 6 shows body weight of Syrian golden hamsters that had consumed ahyperglycemia-inducing diet with and without algal biomass from culturedChlorella protothecoides. Values are mean±SEM and body weight gain wasnot significantly different between groups (repeated measures ANOVA).

FIGS. 7a-c show a 23S rRNA genomic sequence analysis of strainsdesignated as Chlorella protothecoides, as described in the Examplesbelow.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

The term “administering” refers to oral administration, unless otherwiseindicated.

“Axenic” means a culture of an organism that is free from contaminationby other living organisms.

“Bioreactor” means an enclosure or partial enclosure in which cells arecultured, optionally in suspension.

“Cellulosic material” means the products of digestion of cellulose,including glucose and xylose, and optionally additional compounds suchas disaccharides, oligosaccharides, lignin, furfurals and othercompounds. Nonlimiting examples of sources of cellulosic materialinclude sugar caner bagasses, sugar beet pulp, corn stover, wood chips,sawdust and switchgrass.

The term “cofactor” is used herein to refer to any molecule, other thanthe substrate, that is required for an enzyme to carry out its enzymaticactivity.

As used herein, “conventional food product” refers to a compositionintended for consumption, e.g., by a human, that includes typicalingredients that one would ordinary associate with the food product. Forexample, a conventional cake might ordinarily include grain flour, eggs,sugar, butter, milk, and a flavoring extract. Some conventional cakesmight also include fruits, nuts and/or chocolate.

As used herein, “cooked product” refers to a composition that has beenheated, e.g., in an oven, for a period of time.

The term “cultivated”, and variants thereof, refer to the intentionalfostering of growth (increases in cell size, cellular contents, and/orcellular activity) and/or propagation (increases in cell numbers viamitosis or another method of cell division) of one or more cells by useof intended culture conditions. The combination of both growth andpropagation may be termed proliferation. The one or more cells may bethose of a microorganism, such as microalgae. Examples of intendedconditions include the use of a defined medium (with knowncharacteristics such as pH, ionic strength, and carbon source),specified temperature, oxygen tension, carbon dioxide levels, and growthin a bioreactor. The term does not refer to the growth or propagation ofmicroorganisms in nature or otherwise without direct human intervention.

“Diabetes,” as used herein, refers to diabetes mellitus and includestype 1, type 2, and type 3 (also referred to as type 1.5) unlessotherwise indicated. “Diabetes” corresponds to a fasting plasma glucoseconcentration greater than or equal to 126 mg/dl (6.9 mmol/1), or aplasma glucose concentration greater than or equal to 200 mg/dl (11.1mmol/1) two hours after ingestion of a 75 g oral glucose load.

As used herein, the terms “dry weight” or “dry cell weight” refer toweight as determined in the relative absence of water. For example,reference to a component of microalgal biomass as comprising a specifiedpercentage by dry weight means that the percentage is calculated basedon the weight of the biomass after substantially all water has beenremoved.

As used herein, “edible ingredient” refers to any substance orcomposition which is fit to be eaten. “Edible ingredients” include,without limitation, grains, fruits, vegetables, proteins, herbs, spices,carbohydrates, and fats.

“Exogenously provided” describes a molecule provided to the culturemedia of a cell culture.

As used herein, “finished food product,” or “finished food ingredient”refers to a food composition that is ready for packaging, use, orconsumption. For example, a “finished food product” may have been cookedor the ingredients comprising the “finished food product” may have beenmixed or otherwise integrated with one another. A “finished foodproduct” is suitable for consumption. A “finished food ingredient” issuitable for consumption or for combination with other ingredients toform a food product.

As used herein, “food composition” refers to any composition intended tobe or reasonably expected to be ingested by humans as a source ofnutrition and/or calories. Food compositions are composed primarily ofcarbohydrates, fats, water and/or proteins and make up substantially allof a person's daily caloric intake. A “food composition” has a weightminimum that is at least ten (10) times the weight of a typical tabletor capsule (typical tablet weight ranges from 100 mg up to 1500 mg). A“food composition” is not encapsulated or in tablet form.

“Fixed carbon source” means molecule(s) containing carbon, preferablyorganic, that are present at ambient temperature and pressure in solidor liquid form.

“Glycerolipid profile” refers to the distribution of different carbonchain lengths and saturation levels of glycerolipids in a particularsample of biomass or oil. For example, a sample could containglycerolipids in which approximately 60% of the glycerolipid is C18:1,20% is C18:0, 15% is C16:0, and 5% is C14:0. In cases in which a carbonlength is referenced generically, such as “C:18”, such reference caninclude any amount of saturation; for example, microalgal biomass thatcontains 20% lipid as C:18 can include C18:0, C18:1, C18:2, and thelike, in equal or varying amounts, the sum of which constitute 20% ofthe biomass.

As used herein, “good manufacturing practice” or “GMP” conditions referto conditions established by regulations set forth at 21 C.F.R. 110 (forhuman food), 111 (for dietary supplements), and 210-211 (for drugproducts) or comparable regulatory schemes established in localesoutside the United States. The U.S. regulations are promulgated by theU.S. Food and Drug Administration under the authority of the FederalFood, Drug, and Cosmetic Act to regulate manufacturers, processors, andpackagers of drugs, food products and dietary supplements for humanadministration or consumption.

“Homogenate” means biomass that has been physically disrupted.

“Impaired fasting glucose” means a fasting plasma glucose concentrationgreater than or equal to 100 mg/dl (5.6 mmol/1).

“Impaired glucose tolerance” means a plasma glucose concentrationgreater than or equal to 140 mg/dl (7.8 mmol/1) two hours afteringestion of a 75 g oral glucose load.

As used herein, the phrase “increase lipid yield” refers to an increasein the productivity of a microbial culture by, for example, increasingdry weight of cells per liter of culture, increasing the percentage ofcells that constitute lipid, or increasing the overall amount of lipidper liter of culture volume per unit time.

“Lipids” are a class of hydrocarbon that are soluble in nonpolarsolvents (such as ether and chloroform) and are relatively or completelyinsoluble in water. Lipid molecules have these properties because theyconsist largely of long hydrocarbon tails which are hydrophobic innature. Examples of lipids include fatty acids (saturated andunsaturated); glycerides or glycerolipids (such as monoglycerides,diglycerides, triglycerides or neutral fats, and phosphoglycerides orglycerophospholipids); nonglycerides (sphingolipids, sterol lipidsincluding cholesterol and steroid hormones, prenol lipids includingterpenoids, fatty alcohols, waxes, and polyketides); and complex lipidderivatives (sugar-linked lipids, or glycolipids, and protein-linkedlipids). “Fats” and “oils” are also known as triacylglycerides,triglycerides, tryacylglycerols, or glycerolipids.

As used herein, the term “lysate” refers to a solution containing thecontents of lysed cells.

As used herein, the term “lysis” refers to the breakage of the plasmamembrane and optionally the cell wall of a biological organismsufficient to release at least some intracellular content, often bymechanical, viral or osmotic mechanisms that compromise its integrity.The term “lysing” refers to disrupting the cellular membrane andoptionally the cell wall of a biological organism or cell sufficient torelease at least some intracellular content.

The term “mammal” includes, without limitation, humans, domestic animals(e.g., dogs or cats), farm animals (e.g., cows, horses, or pigs),monkeys, rabbits, mice, and laboratory animals.

As used herein, “microalgal biomass,” “algal biomass,” or “biomass”refers to Chlorella protothecoides material produced by growth and/orpropagation of Chlorella protothecoides cells. Biomass may contain cellsand/or intracellular contents as well as extracellular material.Extracellular material includes, but is not limited to, compoundssecreted by a cell.

As used herein, “microalgal oil” or “algal oil” or “oil” refers to lipidcomponents produced by Chlorella protothecoides cells, includingtriacylglycerols.

The terms “microorganism” and “microbe” are used interchangeably hereinto refer to microscopic unicellular organisms.

“Patient” refers to human and non-human animals, especially mammals.Examples of patients include, but are not limited to, humans, cows,dogs, cats, goats, sheep, pigs and rabbits.

The term “pharmaceutically acceptable carrier or excipient” means acarrier or excipient that is useful in preparing a pharmaceuticalcomposition that is generally safe, non-toxic and neither biologicallynor otherwise undesirable, and includes a carrier or excipient that isacceptable for veterinary use as well as human pharmaceutical use. A“pharmaceutically acceptable carrier or excipient” as used in thespecification and claims includes both one and more than one suchcarrier or excipient.

The terms “pharmaceutically effective amount”, “therapeuticallyeffective amount”, “therapeutically effective dose”, or “effectiveregime” refer to the amount of Chlorella protothecoides biomass andschedule of administration that will elicit the desired biological ormedical response of the patient or subject to which the biomass isadministered. The effective amount will vary depending on the disorderor condition and its severity, as well as the age, weight, etc., of thepatient or subject to be treated.

“Photobioreactor” refers to a container, at least part of which is atleast partially transparent or partially open, thereby allowing light topass through, in which one or more microalgae cells are cultured.Photobioreactors may be closed, as in the instance of a polyethylene bagor Erlenmeyer flask, or may be open to the environment, as in theinstance of an outdoor pond.

As used herein, a “polysaccharide-degrading enzyme” refers to any enzymecapable of catalyzing the hydrolysis, or depolymerization, of anypolysaccharide. For example, cellulases catalyze the hydrolysis ofcellulose.

“Polysaccharides” (also called “glycans”) are carbohydrates made up ofmonosaccharides joined together by glycosidic linkages. Cellulose is anexample of a polysaccharide that makes up certain plant cell walls.Cellulose can be depolymerized by enzymes to yield monosaccharides suchas xylose and glucose, as well as larger disaccharides andoligosaccharides.

“Port”, in the context of a bioreactor, refers to an opening in thebioreactor that allows influx or efflux of materials such as gases,liquids, and cells. Ports are usually connected to tubing leading fromthe photobioreactor.

As used herein, “predominantly encapsulated” means that more than 50%and typically more than 75% or 90% of a referenced component, e.g.,algal oil, is sequestered in a referenced container, e.g., a microalgalcell.

As used herein, “predominantly intact cells” refers to a population ofcells which comprise more than 50, 75 or 90% intact cells. “Intact”refers to the physical continuity of the cellular membrane enclosing theintracellular components of the cell and means that the cellularmembrane has not been disrupted in any manner that would release theintracellular components of the cell to an extent that exceeds thepermeability of the cellular membrane under conventional cultureconditions or those culture conditions described herein.

As used herein, “predominantly lysed” refers to a population of cells,of which more than 50%, and often more than 75 or 90% have beendisrupted such that the intracellular components of the cell are nolonger enclosed within the cell membrane.

The terms “prevent”, “preventing”, “prevention” and grammaticalvariations thereof as used herein, refer to a method of partially orcompletely delaying or precluding the onset or recurrence of a disorderor condition and/or one or more of its attendant symptoms or barring asubject from acquiring or reacquiring a disorder or condition orreducing a subject's risk of acquiring or reaquiring a disorder orcondition or one or more of its attendant symptoms.

As used herein, “proximate to a meal” means within a period fromapproximately one hour before beginning a meal, during a meal, or up toapproximately one hour after finishing a meal.

As used herein, “stover” refers to the dried stalks and leaves of a cropremaining after a grain has been harvested.

The “subject” is defined herein to include animals such as mammals,including, but not limited to, primates (e.g., humans), cows, sheep,goats, horses, dogs, cats, rabbits, rats, mice and the like. Inpreferred embodiments, the subject is a human.

The terms “treat”, “treating”, “treatment” and grammatical variationsthereof as used herein, includes partially or completely delaying,alleviating, mitigating or reducing the intensity of one or moreattendant symptoms of a disorder or condition and/or alleviating,mitigating or impeding one or more causes of a disorder or condition.Treatments according to the invention may be applied preventively,prophylactically, pallatively or remedially.

As used herein, “uncooked product” refers to a composition that has notbeen subjected to heating. An “uncooked product” includes a compositionthat contains one or more components that were formerly subjected toheating.

Reference to proportions by volume, i.e., “v/v,” means the ratio of thevolume of one substance or composition to the volume of a secondsubstance or composition. For example, reference to a composition thatcomprises 5% v/v microalgal oil and at least one other edible ingredientmeans that 5% of the composition's volume is composed of microalgal oil;e.g., a composition having a volume of 100 mm³ would contain 5 mm³ ofmicroalgal oil and 95 mm³ of other constituents.

Reference to proportions by weight, i.e., “w/w,” means the ratio of theweight of one substance or composition to the weight of a secondsubstance or composition. For example, reference to a composition thatcomprises 5% w/w microalgal biomass and at least one other edibleingredient means that 5% of the composition's weight is composed ofmicroalgal biomass; e.g., a 100 mg composition would contain 5 mg ofmicroalgal biomass and 95 mg of other constituents.

For sequence comparison to determine percent nucleotide or amino acididentity, typically one sequence acts as a reference sequence, to whichtest sequences are compared. When using a sequence comparison algorithm,test and reference sequences are input into a computer, subsequencecoordinates are designated, if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

Another example algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information.

II. General

The invention provides methods of treating a patient having impairedglucose tolerance via administration of an effective regimen ofChlorella protothecoides biomass comprising at least 15% algal oil bydry weight. The invention also provides methods of reducing bloodglucose and/or body fat in a subject via administration of an effectiveregimen of Chlorella protothecoides biomass, as well as methods ofincreasing the relative abundance of beneficial gut microflora (e.g.,Lactobacillales). Some aspects of the invention are premised in part onthe insight that Chlorella protothecoides biomass can be prepared with ahigh oil content, and that the resulting biomass, incorporated into afood product or other dosage form, can be used to lower blood glucoselevels in a subject independent of the subject's plasma insulinconcentrations. Thus, in some aspects, the methods of the invention canbe used to treat patient's with type 1, type 2, or type 1.5 diabetes.

The administration of Chlorella protothecoides biomass in accordancewith the methods of the invention can be effected, in some cases, bysubstituting the biomass or a biomass-containing dosage form (e.g.,tablets or a food product) for from 1-20% of the food by weight orcalories in a patient's diet.

III. Chlorella Protothecoides

Considerations affecting the selection of microalgae for use in theinvention include, in addition to production of suitable biomass: (1)high lipid content as a percentage of cell weight; (2) ease of growth;and (3) ease of biomass processing. In some embodiments, strains ofmicroalgae having cell walls susceptible to digestion in thegastrointestinal tract of an animal, e.g., a human, are preferred. Thiscriterion is particularly preferred when the algal biomass isadministered in an uncooked form in accordance with the presentinvention. Digestibility is generally decreased for microalgal strainswhich have a high content of cellulose/hemicellulose in the cell walls.Digestibility can be evaluated using a standard pepsin digestibilityassay.

In particular embodiments, the wild-type or genetically engineeredmicroalgae comprise cells that are at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, or at least 80% or more oil by dry weight. Preferred organisms growheterotrophically (on sugars in the absence of light) or can beengineered to do so using, for example, methods disclosed herein. Theease of transformation and availability of selectable markers andpromoters, constitutive and/or inducible, that are functional in themicroalgae affect the ease of genetic engineering. Processingconsiderations can include, for example, the availability of effectivemeans for lysing the cells.

Chlorella protothecoides, when cultured heterotrophically, can containhigher oil content and impart the glucose modulation and prebioticeffects disclosed herein. Chlorella protothecoides is a single-celledgreen algae, belonging to the phylum Chlorophyta. It is spherical inshape, about 2 to 10 μm in diameter, and is without flagella.

Chlorella protothecoides for use in the invention can be identified byamplification of certain target regions of the genome. For example,identification of Chlorella protothecoides can be achieved throughamplification and sequencing of nuclear and/or chloroplast DNA usingprimers and methodology using any region of the genome, for exampleusing the methods described in Wu et al., Bot. Bull. Acad. Sin.42:115-121 (2001). Well established methods of phylogenetic analysis,such as amplification and sequencing of ribosomal internal transcribedspacer (ITS1 and ITS2 rDNA), 18S rRNA, 23S rRNA, and other conservedgenomic regions can be used by those skilled in the art to identifyspecies and strain. For general examples of methods of identificationand classification of algae see Genetics, 170(4):1601-10 (2005) and RNA,11(4):361-4 (2005).

Genomic DNA comparison can also be useful to identify Chlorellaprotothecoides strains that have been misidentified in a straincollection. Often a strain collection will identify species ofmicroalgae based on phenotypic and morphological characteristics. Theuse of these characteristics may lead to miscategorization of thespecies or genus of a microalgae. The use of genomic DNA comparison canbe a better method of identifying Chlorella protothecoides strains thatare suitable for use in the present invention. Specific examples usinggenotyping data to establish phylogenetic relationship/identity foreight Chlorella protothecoides strains from different collections aredescribed below in the Examples.

In some cases, microalgae that are preferred for use in the presentinvention have genomic DNA sequences encoding for 23S rRNA that have nomore than 1 base pair, no more than 2 base pairs, no more than 3 basepairs, no more than 4 base pairs, no more than 5 base pairs, no morethan 6 base pairs, no more than 7 base pairs, no more than 8 base pairs,no more than 9 base pairs or no more than 10 base pairs difference tothat of the sequences listed in SEQ ID NO: 5 or SEQ ID NO:6.

IV. Methods of Culturing Chlorella Protothecoides

Chlorella protothecoides can be cultured for production of algalbiomass. Culture for purposes of biomass production is usually conductedon a large scale. Preferably a fixed carbon source is present. Theculture can also be exposed to light some or all of the time.

Chlorella protothecoides can be cultured in liquid media. The culturecan be contained within a bioreactor. Optionally, the bioreactor doesnot allow light to enter. Alternatively, cells can also be cultured inphotobioreactors that contain a fixed carbon source and allow light tostrike the cells. Culture condition parameters can be manipulated tooptimize total oil production, the combination of lipid speciesproduced, and/or production of a specific oil. In some instances it ispreferable to culture cells in the dark, such as, for example, whenusing extremely large (40,000 liter and higher) fermentors that do notallow light to strike the culture.

Culture media typically contains components such as a fixed nitrogensource, trace elements, optionally a buffer for pH maintenance, andphosphate. Other components can include a fixed carbon source such asacetate or glucose, and salts such as sodium chloride, particularly forseawater microalgae. Examples of trace elements include zinc, boron,cobalt, copper, manganese, and molybdenum in, for example, therespective forms of ZnCl₂, H₃BO₃, CoCl₂.6H₂O, CuCl₂.2H₂O, MnCl₂.4H₂O and(NH₄)₆Mo₇O₂₄.4H₂O.

For organisms able to grow on a fixed carbon source, the fixed carbonsource can be, for example, glucose, fructose, sucrose, galactose,xylose, mannose, rhamnose, N-acetylglucosamine, glycerol, floridoside,glucuronic acid, and/or acetate. The one or more carbon source(s) can besupplied at a concentration of at least about 50 μM, at least about 100μM, at least about 500 μM, at least about 5 mM, at least about 50 mM,and at least about 500 mM, of one or more exogenously provided fixedcarbon source(s). Some microalgae species can grow by utilizing a fixedcarbon source such as glucose or acetate in the absence of light. Suchgrowth is known as heterotrophic growth. For Chlorella protothecoides,for example, heterotrophic growth results in high production of biomassand accumulation of high lipid content in cells.

Other culture parameters can also be manipulated, such as the pH of theculture media, the identity and concentration of trace elements, andother media constituents.

A. Photosynthetic Growth

Microalgae can be grown in the presence of light. The number of photonsstriking a culture of microalgae cells can be manipulated, as well asother parameters such as the wavelength spectrum and ratio of dark:lighthours per day. Microalgae can also be cultured in natural light, as wellas simultaneous and/or alternating combinations of natural light andartificial light. For example, microalgae of the genus Chlorella can becultured under natural light during daylight hours and under artificiallight during night hours.

The gas content of a photobioreactor to grow microorganisms likemicroalgae can be manipulated. Part of the volume of a photobioreactorcan contain gas rather than liquid. Gas inlets can be used to pump gasesinto the photobioreactor. Any gas can be pumped into a photobioreactor,including air, air/CO₂ mixtures, noble gases such as argon and others.The rate of entry of gas into a photobioreactor can also be manipulated.Increasing gas flow into a photobioreactor increases the turbidity of aculture of microalgae. Placement of ports conveying gases into aphotobioreactor can also affect the turbidity of a culture at a givengas flow rate. Air/CO₂ mixtures can be modulated to generate optimalamounts of CO₂ for maximal growth by a particular organism. Microalgaegrow significantly faster in the light under, for example, 3% CO₂/97%air than in 100% air. 3% CO₂/97% air is approximately 100-fold more CO₂than found in air. For example, air:CO₂ mixtures of about 99.75%air:0.25% CO₂, about 99.5% air:0.5% CO₂, about 99.0% air:1.00% CO₂,about 98.0% air:2.0% CO₂, about 97.0% air:3.0% CO₂, about 96.0% air:4.0%CO₂, and about 95.00% air:5.0% CO₂ can be infused into a bioreactor orphotobioreactor.

Microalgae cultures can also be subjected to mixing using devices suchas spinning blades and impellers, rocking of a culture, stir bars,infusion of pressurized gas, and other instruments.

Photobioreactors can have ports allowing entry of gases, solids,semisolids and liquids into the chamber containing the microalgae. Portsare usually attached to tubing or other means of conveying substances.Gas ports, for example, convey gases into the culture. Pumping gasesinto a photobioreactor can serve to both feed cells CO₂ and other gasesand to aerate the culture and therefore generate turbidity. The amountof turbidity of a culture varies as the number and position of gas portsis altered. For example, gas ports can be placed along the bottom of acylindrical polyethylene bag. Microalgae grow faster when CO₂ is addedto air and bubbled into a photobioreactor.

Photobioreactors can be exposed to one or more light sources to providemicroalgae with light as an energy source via light directed to asurface of the photobioreactor. Preferably the light source provides anintensity that is sufficient for the cells to grow, but not so intenseas to cause oxidative damage or cause a photoinhibitive response. Insome instances a light source has a wavelength range that mimics orapproximately mimics the range of the sun. In other instances adifferent wavelength range is used. Photobioreactors can be placedoutdoors or in a greenhouse or other facility that allows sunlight tostrike the surface, such as the use of Chlorella protothecoides inphotobioreactors described in U.S. patent 20080160591.

Photobioreactors preferably have one or more ports that allow mediaentry. It is not necessary that only one substance enter or leave aport. For example, a port can be used to flow culture media into thephotobioreactor and then later can be used for sampling, gas entry, gasexit, or other purposes. In some instances a photobioreactor is filledwith culture media at the beginning of a culture and no more growthmedia is infused after the culture is inoculated. In other words, themicroalgal biomass is cultured in an aqueous medium for a period of timeduring which the microalgae reproduce and increase in number; howeverquantities of aqueous culture medium are not flowed through thephotobioreactor throughout the time period. Thus in some embodiments,aqueous culture medium is not flowed through the photobioreactor afterinoculation.

In other instances culture media can be flowed though thephotobioreactor throughout the time period during which the microalgaereproduce and increase in number. In some embodiments media is infusedinto the photobioreactor after inoculation but before the cells reach adesired density. In other words, a turbulent flow regime of gas entryand media entry is not maintained for reproduction of microalgae until adesired increase in number of said microalgae has been achieved.

Photobioreactors preferably have one or more ports that allow gas entry.Gas can serve to both provide nutrients such as CO₂ as well as toprovide turbulence in the culture media. Turbulence can be achieved byplacing a gas entry port below the level of the aqueous culture media sothat gas entering the photobioreactor bubbles to the surface of theculture. One or more gas exit ports allow gas to escape, therebypreventing pressure buildup in the photobioreactor. Preferably a gasexit port leads to a “one-way” valve that prevents contaminatingmicroorganisms from entering the photobioreactor. In some instancescells are cultured in a photobioreactor for a period of time duringwhich the microalgae reproduce and increase in number, however aturbulent flow regime with turbulent eddies predominantly throughout theculture media caused by gas entry is not maintained for all of theperiod of time. In other instances a turbulent flow regime withturbulent eddies predominantly throughout the culture media caused bygas entry can be maintained for all of the period of time during whichthe microalgae reproduce and increase in number. In some instances apredetermined range of ratios between the scale of the photobioreactorand the scale of eddies is not maintained for the period of time duringwhich the microalgae reproduce and increase in number. In otherinstances such a range can be maintained.

Photobioreactors preferably have at least one port that can be used forsampling the culture. Preferably a sampling port can be used repeatedlywithout altering compromising the axenic nature of the culture. Asampling port can be configured with a valve or other device that allowsthe flow of sample to be stopped and started. Alternatively a samplingport can allow continuous sampling. Photobioreactors preferably have atleast one port that allows inoculation of a culture. Such a port canalso be used for other purposes such as media or gas entry.

B. Heterotrophic Growth

As an alternative to photosynthetic growth of microorganisms, asdescribed above, some microorganisms can be cultured under heterotrophicgrowth conditions in which a fixed carbon source provides energy forgrowth and lipid accumulation. In some cases, the fixed carbon energysource comprises cellulosic material, including depolymerized cellulosicmaterial, a 5-carbon sugar, or a 6-carbon sugar.

Standard methods for the heterotrophic growth and propagation ofChlorella protothecoides are known (see for example Miao and Wu, J.Biotechnology, 2004, 11:85-93). Under certain heterotrophic conditionswith nitrogen starvation, Chlorella protothecoides can produce up to 55%lipid (as measured by dry cell weight (DCW) (Miao and Wu, BiosourceTechnology (2006) 97:841-846). Glucose and nitrogen conditions are alsoimportant for the generation of increased biomass and lutein inChlorella protothecoides (Shi et al., Enzy Microbio Tech, (2000)27:312-318 and Shi et al., Process Biochem, (1999) 34: 341-347). Theinvention also provides novel growth conditions for Chlorella. Forexample, multiple strains of Chlorella protothecoides can be grown inthe presence of glycerol. Examples below describe specific cultureconditions of Chlorella protothecoides grown on glycerol as a carbonsource. FIG. 1 shows high lipid accumulation (DCW) in Chlorellaprotothecoides when grown on a combination of glucose and glycerol. Thepercent of dry cell weight as lipid can be modulated by the length oftime the cells are cultured in a given culture media that is repletewith a fixed carbon source under conditions of limited nitrogen. Ingeneral, the longer the cells are held in such conditions the higher thepercent lipid as dry cell weight becomes.

The invention provides significantly improved culture parametersincorporating the use of glycerol for fermentation of multiple genera ofmicroalgae. As the Examples demonstrate, many Chlorella protothecoidesstrains grow very well on not only purified reagent-grade glycerol, butalso on acidulated and non-acidulated glycerol byproduct from biodieseltransesterification. In some instances, microalgae, such as Chlorellastrains, undergo cell division faster in the presence of glycerol thanin the presence of glucose. In these instances, two-stage growthprocesses in which cells are first fed glycerol to rapidly increase celldensity, and are then fed glucose to accumulate lipids can improve theefficiency with which lipids are produced.

Other feedstocks for culturing microalgae in accordance with the presentinvention are provided as well, such as mixtures of glycerol andglucose, mixtures of glucose and xylose, mixtures of fructose andglucose, sucrose, glucose, fructose, xylose, arabinose, mannose,galactose, acetate, and molasses. Also provided are methods utilizingcorn stover, sugar beet pulp, and switchgrass in combination withdepolymerization enzymes. The use of these alternative feedstocks isdemonstrated in the Examples provided herein.

For lipid and oil production, cells, including recombinant cells of theinvention described herein, are preferably cultured or fermented inlarge quantities. The culturing may be in large liquid volumes, such asin suspension cultures as an example. Other examples include startingwith a small culture of cells which expand into a large biomass incombination with cell growth and propagation as well as lipid and oilproduction. Bioreactors or steel fermentors can be used to accommodatelarge culture volumes. A fermentor similar those used in the productionof beer and/or wine is suitable, as are extremely large fermentors usedin the production of ethanol.

Appropriate nutrient sources for culture in a fermentor are provided.These include raw materials such as one or more of the following: afixed carbon source such as glucose, corn starch, depolymerizedcellulosic material, sucrose, sugar cane, sugar beet, lactose, milkwhey, molasses, or the like; a fat source, such as fats or vegetableoils; a nitrogen source, such as protein, soybean meal, cornsteepliquor, ammonia (pure or in salt form), nitrate or nitrate salt, ormolecular nitrogen; and a phosphorus source, such as phosphate salts.Additionally, a fermentor allows for the control of culture conditionssuch as temperature, pH, oxygen tension, and carbon dioxide levels.Optionally, gaseous components, like oxygen or nitrogen, can be bubbledthrough a liquid culture. Other starch (glucose) sources such as wheat,potato, rice, and sorghum. Other carbon sources include process streamssuch as technical grade glycerol, black liquor, organic acids such asacetate, and molasses. Carbon sources can also be provided as a mixture,such as a mixture of sucrose and depolymerized sugar beet pulp.

A fermentor can be used to allow cells to undergo the various phases oftheir growth cycle. As an example, an inoculum of lipid-producing cellscan be introduced into a medium followed by a lag period (lag phase)before the cells begin growth. Following the lag period, the growth rateincreases steadily and enters the log, or exponential, phase. Theexponential phase is in turn followed by a slowing of growth due todecreases in nutrients and/or increases in toxic substances. After thisslowing, growth stops, and the cells enter a stationary phase or steadystate, depending on the particular environment provided to the cells.

Oil production by cells disclosed herein can occur during the log phaseor thereafter, including the stationary phase wherein nutrients aresupplied, or still available, to allow the continuation of oil and lipidproduction in the absence of cell division.

Preferably, microorganisms grown using conditions described herein andknown in the art comprise at least about 15% by dry weight of lipid,preferably at least about 25% to about 35% by dry weight, morepreferably at least about 45% by dry weight, and most preferably atleast about 55% by dry weight.

FIG. 1 demonstrates three different strains of Chlorella protothecoidesaccumulating higher dry cell weight per liter of culture when glyceroland glucose are added sequentially than when the same quantities ofglycerol and glucose are added together at the beginning of theexperiment. This trend was observed when acidulated biodiesel byproductglycerol, non-acidulated biodiesel byproduct glycerol, or reagent gradeglycerol was used.

Three different markers of productivity (dry cell weight per liter,grams per liter of lipid, and percentage of dry cell weight as lipid) inmicrobial lipid production are improved by the use of biodieselbyproduct and temporal separation of carbon sources. The inventiontherefore provides novel methods of generating higher quantities oflipid per unit time in multiple species of microbes from highlydivergent areas of the evolutionary tree, including both prokaryotes andeukaryotes. The methods of manufacturing lipids and oils disclosedherein using glycerol are not limited to microalgae, but can be usedwith any microbe capable of utilizing glycerol as an energy source.

In an alternate heterotrophic growth method in accordance with thepresent invention, microorganisms can be cultured using depolymerizedcellulosic biomass as a feedstock. Cellulosic biomass (e.g., stover,such as corn stover) is inexpensive and readily available; however,attempts to use this material as a feedstock for yeast have failed. Inparticular, such feedstock have been found to be inhibitory to yeastgrowth, and yeast cannot use the 5-carbon sugars produced fromcellulosic materials (e.g., xylose from hemicellulose). By contrast,microalgae can grow on processed cellulosic material. Accordingly, theinvention provides a method of culturing a microalgae in the presence ofa cellulosic material and/or a 5-carbon sugar. Cellulosic materialsgenerally include:

Component Percent Dry Weight Cellulose 40-60% Hemicellulose 20-40%Lignin 10-30%

Suitable cellulosic materials include residues from herbaceous and woodyenergy crops, as well as agricultural crops, i.e., the plant parts,primarily stalks and leaves, not removed from the fields with theprimary food or fiber product. Examples include agricultural wastes suchas sugarcane bagasse, rice hulls, corn fiber (including stalks, leaves,husks, and cobs), wheat straw, rice straw, sugar beet pulp, citrus pulp,citrus peels; forestry wastes such as hardwood and softwood thinnings,and hardwood and softwood residues from timber operations; wood wastessuch as saw mill wastes (wood chips, sawdust) and pulp mill waste; urbanwastes such as paper fractions of municipal solid waste, urban woodwaste and urban green waste such as municipal grass clippings; and woodconstruction waste. Additional cellulosics include dedicated cellulosiccrops such as switchgrass, hybrid poplar wood, and miscanthus, fibercane, and fiber sorghum. Five-carbon sugars that are produced from suchmaterials include xylose.

Surprisingly, Chlorella protothecoides have been shown herein to exhibithigher levels of productivity when cultured on a combination of glucoseand xylose than when cultured on either glucose or xylose alone. Thissynergistic effect provides a significant advantage in that it allowscultivation of Chlorella protothecoides on combinations of xylose andglucose, such as cellulosic material, and is shown in FIG. 2.

In still another alternative heterotrophic growth method in accordancewith the present invention, which itself may optionally be used incombination with the methods described above, sucrose, produced byexample from sugar cane or sugar beet, is used as a feedstock.

In still another alternative heterotrophic growth method in accordancewith the present invention, which itself may optionally be used incombination with the methods described above, sucrose, produced byexample from sugar cane or sugar beet, is used as a feedstock. Somemicroalgae may need an exogenous sucrose utilization enzyme, such as asucrose invertase, to be added to the culture medium in order to utilizethe sucrose as a carbon source.

Bioreactors can be employed for use in heterotrophic growth methods. Aswill be appreciated, provisions made to make light available to thecells in photosynthetic growth methods are unnecessary when using afixed-carbon source in the heterotrophic growth methods describedherein.

The specific examples of process conditions and heterotrophic growthmethods described herein can be combined in any suitable manner toimprove efficiencies of microbial growth and lipid production. Inaddition, the invention includes the selection and/or geneticengineering of microbes, such as microalgae, to produce microbes thatare even more suitable for use in the above-described methods. Forexample, the microbes having a greater ability to utilize any of theabove-described feedstocks for increased proliferation and/or lipidproduction are within the scope of the invention.

C. Mixotrophic Growth

Mixotrophic growth is the use of both light and fixed carbon source(s)as energy sources for cells to grow and produce oils. Mixotrophic growthcan be conducted in a photobioreactor. Microalgae can be grown andmaintained in closed photobioreactors made of different types oftransparent or semitransparent material. Such material can includePlexiglas® enclosures, glass enclosures, bags made from substances suchas polyethylene, transparent or semitransparent pipes, and othermaterials. Microalgae can be grown and maintained in openphotobioreactors such as raceway ponds, settling ponds, and othernon-enclosed containers.

D. Growth Media

Microorganisms useful in accordance with the methods of the presentinvention are found in various locations and environments throughout theworld. As a consequence of their isolation from other species and theirresulting evolutionary divergence, the particular growth medium foroptimal growth and generation of oil and/or lipid can be difficult topredict. In some cases, certain strains of microorganisms may be unableto grow on a particular growth medium because of the presence of someinhibitory component or the absence of some essential nutritionalrequirement required by the particular strain of microorganism.

Solid and liquid growth media are generally available from a widevariety of sources, and instructions for the preparation of particularmedia that is suitable for a wide variety of strains of microorganismscan be found, for example, online at http://www.utex.org/, a sitemaintained by the University of Texas at Austin for its culturecollection of algae (UTEX). For example, various fresh water and saltwater media include those shown in Table 1, below.

TABLE 1 Exemplary Algal Media. Fresh Water Media Salt Water Media 1/2CHEV Diatom Medium 1% F/2 1/3 CHEV Diatom Medium 1/2 Enriched SeawaterMedium 1/5 CHEV Diatom Medium 1/2 Erdschreiber Medium 1:1 DYIII/PEA +Gr+ 1/2 Soil + Seawater Medium 2/3 CHEV Diatom Medium 1/3 Soil +Seawater Medium 2X CHEV Diatom Medium 1/4 ERD Ag Diatom Medium 1/4Soil + Seawater Medium Allen Medium 1/5 Soil + Seawater Medium BG11-1Medium 2/3 Enriched Seawater Medium Bold 1NV Medium 20% Allen + 80% ERDBold 3N Medium 2X Erdschreiber's Medium Botryococcus Medium 2X Soil +Seawater Medium Bristol Medium 5% F/2 Medium CHEV Diatom Medium 5/3Soil + Seawater Agar Medium Chu's Medium Artificial Seawater Medium CR1Diatom Medium BG11-1 + .36% NaCl Medium CR1+ Diatom Medium BG11-1 + 1%NaCl Medium CR1-S Diatom Medium Bold 1NV:Erdshreiber (1:1) CyanidiumMedium Bold 1NV:Erdshreiber (4:1) Cyanophycean Medium Bristol-NaClMedium Desmid Medium Dasycladales Seawater Medium DYIII Medium EnrichedSeawater Medium Euglena Medium Erdschreiber's Medium HEPES Medium ES/10Enriched Seawater Medium J Medium ES/2 Enriched Seawater Medium MaltMedium ES/4 Enriched Seawater Medium MES Medium F/2 Medium Modified Bold3N Medium F/2 + NH4 Modified COMBO Medium LDM Medium N/20 MediumModified 2 X CHEV Ochromonas Medium Modified 2 X CHEV + Soil P49 MediumModified Artificial Seawater Medium Polytomella Medium Modified CHEVProteose Medium Porphridium Medium Snow Algae Media Soil + SeawaterMedium Soil Extract Medium SS Diatom Medium Soilwater: BAR MediumSoilwater: GR− Medium Soilwater: GR−/NH4 Medium Soilwater: GR+ MediumSoilwater: GR+/NH4 Medium Soilwater: PEA Medium Soilwater: Peat MediumSoilwater: VT Medium Spirulina Medium Tap Medium Trebouxia MediumVolvocacean Medium Volvocacean-3N Medium Volvox Medium Volvox-DextroseMedium Waris Medium Waris + Soil Extract Medium

In a particular example, a medium suitable for culturing Chlorellaprotothecoides (UTEX 31) comprises Proteose Medium. This medium issuitable for axenic cultures, and a 1 L volume of the medium (pH˜6.8)can be prepared by addition of 1 g of proteose peptone to 1 liter ofBristol Medium. Bristol medium comprises 2.94 mM NaNO₃, 0.17 mMCaCl₂.2H₂O, 0.3 mM MgSO₄.7H₂O, 0.43 mM, 1.29 mM KH₂PO₄, and 1.43 mM NaClin an aqueous solution. For 1.5% agar medium, 15 g of agar can be addedto 1 L of the solution. The solution is covered and autoclaved, and thenstored at a refrigerated temperature prior to use.

Other suitable media for use with the methods of the invention can bereadily identified by consulting the URL identified above, or byconsulting other organizations that maintain cultures of microorganisms,such as SAG, CCAP, or CCALA. SAG refers to the Culture Collection ofAlgae at the University of Gottingen (Gottingen, Germany), CCAP refersto the culture collection of algae and protozoa managed by the ScottishAssociation for Marine Science (Scotland, United Kingdom), and CCALArefers to the culture collection of algal laboratory at the Institute ofBotany (Tr{hacek over (e)}bo{hacek over (n)}, Czech Republic).

E. Increasing Yield of Lipids

Process conditions can be adjusted to increase the yield of lipidssuitable for a particular use and/or to reduce production cost. Forexample, in certain embodiments, a microbe (e.g., a microalgae) iscultured in the presence of a limiting concentration of one or morenutrients, such as, for example, carbon and/or nitrogen, phosphorous, orsulfur, while providing an excess of fixed carbon energy such asglucose. Nitrogen limitation tends to increase microbial lipid yieldover microbial lipid yield in a culture in which nitrogen is provided inexcess. In particular embodiments, the increase in lipid yield is atleast about: 10%, 20%, 30%, 40%, 50%, 75%, 100%, 200%, 300%, 400%, or500%. The microbe can be cultured in the presence of a limiting amountof a nutrient for a portion of the total culture period or for theentire period. In particular embodiments, the nutrient concentration iscycled between a limiting concentration and a non-limiting concentrationat least twice during the total culture period.

To increase lipid yield, acetic acid can be employed in the feedstockfor a lipid-producing microbe (e.g., a microalgae). Acetic acid feedsdirectly into the point of metabolism that initiates fatty acidsynthesis (i.e., acetyl-CoA); thus providing acetic acid in the culturecan increase fatty acid production. Generally, the microbe is culturedin the presence of a sufficient amount of acetic acid to increasemicrobial lipid yield, and/or microbial fatty acid yield, specifically,over microbial lipid (e.g., fatty acid) yield in the absence of aceticacid.

In another embodiment, lipid yield is increased by culturing alipid-producing microbe (e.g., microalgae) in the presence of one ormore cofactor(s) for a lipid pathway enzyme (e.g., a fatty acidsynthetic enzyme). Generally, the concentration of the cofactor(s) issufficient to increase microbial lipid (e.g., fatty acid) yield overmicrobial lipid yield in the absence of the cofactor(s). In a particularembodiment, the cofactor(s) are provided to the culture by including inthe culture a microbe (e.g., microalgae) containing an exogenous geneencoding the cofactor(s). Alternatively, cofactor(s) may be provided toa culture by including a microbe (e.g., microalgae) containing anexogenous gene that encodes a protein that participates in the synthesisof the cofactor. In certain embodiments, suitable cofactors include anyvitamin required by a lipid pathway enzyme, such as, for example: biotinor pantothenate. Genes encoding cofactors suitable for use in theinvention or that participate in the synthesis of such cofactors arewell known and can be introduced into microbes (e.g., microalgae), usingconstructs and techniques such as those described herein.

V. Methods of Preparing Microalgal Biomass from Algal Cultures

Chlorella protothecoides cultures generated according to the methodsdescribed herein yield microalgal biomass suitable for use in themethods and compositions of the present invention. At the point ofharvesting the microalgal biomass from the culture, the biomasscomprises predominately intact cells suspended in an aqueous culturemedium. The culture medium can be drained off or the biomass otherwiseremoved from the culture medium and subjected to further optionalprocessing, as described below. Optionally, separation of the biomassfrom the culture medium can be effected by centrifugation to generate aconcentrated paste comprising the cells. Centrifugation does not removesignificant amounts of intracellular water. Microalgal biomass can beprocessed to produce a powder, flakes, or a vacuum-packed cake, inexemplary embodiments.

The processes described below can be performed according to GMPconditions in accordance with applicable U.S. or foreign regulations.

A. Drying Biomass

In some cases, drying the microalgal biomass is advantageous tofacilitate further processing or for use of the biomass in the methodsand compositions described herein. Drying the biomass generated from thecultured microalgae described herein removes water that may be anundesirable component of the dosage forms described herein. Drying thebiomass is an optional process step.

In some cases, the biomass can be dried using a drum dryer in which thebiomass is rotated in a drum and dried with the application of air,which may be heated to expedite the drying process. In other cases, anoven or spray drying can be used to facilitate drying of the biomass.Alternatively, the biomass may be dried via a lyophilization process.The lyophilization process can summarily be described as a“freeze-drying” process, in which the biomass is frozen in afreeze-drying chamber to which a vacuum is applied. The application of avacuum to the freeze-drying chamber results in sublimation (primarydrying) and desorption (secondary drying) of the water from the biomass,resulting in a product for further processing in accordance with themethods described herein.

B. Lysing Cells

In those instances in which biomass comprising other than predominantlyintact cells is desired for use in the methods or compositions of thepresent invention, an optional process step of lysing the cells can beperformed. When the cells have been cultured for a desired period or toa desired density, and separated from the culture medium, the cells maybe lysed to provide a homogenated biomass or to facilitate extraction ofmicroalgal oil.

In some cases, the biomass is washed with a washing solution (e.g., DIwater) to get rid of the fermentation broth and debris prior to celldisruption. Optionally, the washed microbial biomass may also be dried(oven dried, lyophilized, etc.) prior to cell disruption. Alternatively,cells can be lysed without separation from some or all of thefermentation broth when the fermentation is complete. For example, thecells can be at a ratio of less than 1:1 v:v cells to extracellularliquid when the cells are lysed.

Microalgae containing lipids can be lysed to produce a lysate. The stepof lysing a microorganism (also referred to as cell lysis) can beachieved by any convenient means, including heat-induced lysis, adding abase, adding an acid, using enzymes such as proteases and polysaccharidedegradation enzymes such as amylases, using ultrasound, mechanicallysis, using osmotic shock, infection with a lytic virus, and/orexpression of one or more lytic genes. Lysis is performed to releaseintracellular molecules which have been produced by the microorganism.Each of these methods for lysing a microorganism can be used as a singlemethod or in combination simultaneously or sequentially.

The extent of cell disruption can be observed by microscopic analysis.Using one or more of the methods described herein, typically more than70% cell breakage is observed. Preferably, cell breakage is more than80%, more preferably more than 90% and most preferred about 100%.

C. GMP Conditions

The processes described herein can be performed in accordance with GMPregulations. In the United States, GMP regulations for manufacturing,processing, packaging and holding drug products for administration tohumans or animals are codified at 21 CFR 210-211. In some embodiments inaccordance with the invention, microalgal biomass can be administered inthe form of a food product. As applicable, GMP regulations formanufacturing, packing, or holding human food are codified at 21 CFR110. The provisions related to drugs and food products, including allparts thereof, as well as ancillary provisions referenced therein, arehereby incorporated by reference in their entirety for all purposes.

In the context of drug products, GMP conditions in the United States orin other jurisdictions include adherence to regulations setting forthquality control requirements for manufacturing, processing, packagingand holding drug products, as well as providing qualification andresponsibility requirements for personnel engaged in these activities,and for the design and maintenance of facilities and equipment used inthe performance of these activities, among other things.

In the context of food products, GMP conditions apply in determiningwhether a food is adulterated in that the food has been manufacturedunder such conditions that it is unfit for food, or in that the food hasbeen prepared, packed, or held under insanitary conditions whereby itmay have become contaminated with filth, or whereby it may have beenrendered injurious to health. GMP conditions can include adherence toregulations governing: disease control, cleanliness and training ofpersonnel; maintenance and sanitary operation of facilities andequipment; provision of appropriate quality control procedures toprevent contamination from any source; and storage and transportation offinished food under conditions that will protect food against physical,chemical, or undesirable microbial contamination, as well as againstdeterioration of the food and the container.

VI. Composition of Microalgal Biomass and Algal Oil

The microalgal biomass generated by the culture methods described hereincomprises microalgal oil as well as other constituents generated by themicroorganisms or incorporated by the microorganisms from the culturemedium during fermentation.

A. Oil Content

Chlorella protothecoides microalgal biomass generated by the culturemethods described herein and useful in accordance with the presentinvention comprises at least 15% microalgal oil by dry weight. In someembodiments, the microalgal biomass comprises at least 25%, at least35%, at least 45%, at least 50%, at least 55%, or at least 60%microalgal oil by dry weight. In some embodiments, the microalgalbiomass contains from 15-90% microalgal oil, from 25-75% microalgal oil,from 40-75% microalgal oil, or from 50-70% microalgal oil by dry weight.

In various embodiments, the microalgal biomass comprises at least 16%,at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, atleast 22%, at least 23%, at least 24%, at least 25%, at least 26%, atleast 27%, at least 28%, at least 29%, at least 30%, at least 35%, atleast 36%, at least 37%, at least 38%, at least 39%, at least 40%, atleast 41%, at least 42%, at least 43%, at least 44%, at least 45%, atleast 46%, at least 47%, at least 48%, at least 49%, or at least 50%microalgal oil by dry weight. In other embodiments, the microalgalbiomass comprises at least 51%, at least 52%, at least 53%, at least54%, at least 55%, at least 56%, at least 57%, at least 58%, at least59%, at least 60%, at least 61%, at least 62%, at least 63%, at least64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, or at least 90% microalgal oil by dry weight.

B. Oil Composition

The oil of the Chlorella protothecoides biomass described herein for usein the methods and compositions of the present invention can compriseglycerolipids with one or more distinct fatty acid ester side chains.Glycerolipids are comprised of a glycerol molecule esterified to one,two, or three fatty acid molecules, which can be of varying lengths andhave varying degrees of saturation.

In some embodiments, the microalgal oil is primarily comprised ofmonounsaturated oil. In some cases, the algal oil is at least 50%monounsaturated oil by weight. In various embodiments, the algal oil isat least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, or at least 80% or more monounsaturated oil by weight or byvolume. In some embodiments, the microalgal oil comprises at least 10%,at least 20%, at least 30%, at least 40%, or at least 50% or moreesterified oleic acid or esterified alpha-linolenic acid by weight of byvolume. The algal oil comprises less than 1% by weight or by volume, oris substantially free of, esterified docosahexanoic acid (DHA).

In some cases, the microalgal biomass comprises algal oil predominantlyencapsulated in cells of the biomass. In other cases, the biomasscomprises predominantly lysed cells and the algal oil is thus primarilynot encapsulated in microalgal cells.

C. Other Constituents

Chlorella protothecoides microalgal biomass can also include otherconstituents produced by the microalgae, or incorporated into themicroalgae from the culture medium. These other constituents can bepresent in varying amounts.

The other constituents can include, without limitation, phospholipids(e.g., algal lecithin), carbohydrates, glycoproteins, phytosterols(e.g., β-sitosterol, campesterol, stigmasterol, ergosterol, andbrassicasterol), tocopherols, tocotrienols, carotenoids (e.g.,α-carotene, β-carotene, and lycopene), xanthophylls (e.g., lutein,zeaxanthin, α-cryptoxanthin, and β-cryptoxanthin), proteins,polysaccharides, and various organic or inorganic compounds (e.g.,selenium).

VII. Methods of Treating Impaired Glucose Metabolism

In one aspect, the present invention is directed to a method of treatinga patient having impaired glucose metabolism. In other embodiments, thepatient has a condition such as impaired glucose tolerance, dysglycemia,insulin resistance, cardiovascular disease, diabetes, hyperglycemia,insulin deficiency, and/or metabolic syndrome. In these and otherembodiments, the methods involve first diagnosing that the patient is inneed of treatment (i.e., exhibits a symptom of one or more of thefollowing conditions), and then, administering an effective regime ofChlorella protothecoides biomass such that the symptom(s) lessen or goaway completely. In one embodiment, the method comprises administeringto the patient an effective regime of Chlorella protothecoides biomasscomprising at least 15% algal oil by dry weight. In some cases, methodsof the invention comprise reducing blood glucose levels in a subjectrelative to a level prior to treatment with the algal biomass regime. Inother cases, methods of the invention comprise reducing the percentagefat of total body weight of the subject relative to the percentage fatof total body weight before administering the algal biomass regime.

In some cases, a patient's impaired glucose metabolism is identified asimpaired glucose tolerance. As defined herein, impaired glucosetolerance corresponds to a glucose concentration of 140 mg/dl (7.8mmol/1) or more, as measured by an oral glucose tolerance test (OGTT).An OGTT is a routine assay used to assess an individual's capacity tometabolize a bolus of glucose. Typically, the patient ingests a 75 gglucose load, and an assessment of plasma glucose concentration is made2 hours after ingestion.

In other cases, a patient's impaired glucose metabolism is identified asimpaired fasting glucose. As defined herein, impaired fasting glucosecorresponds to a glucose concentration of 100 mg/dl (5.6 mmol/1) ormore, as measured by a fasting plasma glucose test (FPGT). An FPGT is aroutine assay used to assess an individual's plasma glucoseconcentration following a fasting period of at least 8 hours.

In still other cases, a patient's impaired glucose metabolism isidentified as diabetes mellitus. A diagnosis of diabetes may be madewith reference to a FPGT or an OGTT, or via other criteria as determinedby a patient's physician. Diabetes mellitus includes types 1, 2, and 3(also referred to as type 1.5). With reference to the assays identifiedabove, diabetes corresponds to a plasma glucose concentration greaterthan or equal to 126 mg/dl (6.9 mmol/1) in the FPGT, or a plasma glucoseconcentration greater than or equal to 200 mg/dl (11.1 mmol/1) in theOGTT.

In those instances in which a patient's glucose concentration exceedsthe normal level (i.e., 99 mg/dl for a FPGT, or 125 mg/dl for an OGTT),but is not high enough to be classified as diabetes, the patient may bereferred to as having a condition known as pre-diabetes. Left untreated,in many instances individuals with pre-diabetes will develop non-insulindependent diabetes. See Keen et al., Diabetologia 22:73-78 (1982).

In some cases, as demonstrated in Example 9, administration of algalbiomass in accordance with the present invention results in a reductionin the mean plasma glucose concentration of a mammal relative to theconcentration before administration of the algal biomass. In some cases,the mean plasma glucose concentration is lowered 10-50%. In some cases,the mean plasma glucose concentration is lowered 10-40%, 20-40%, 30-40%,or 40-50%. In some cases, the reduction in mean plasma glucoseconcentration is at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, orat least 50%. In some cases, the mean plasma glucose concentration islowered from above the diabetic threshold to below the diabeticthreshold or below the pre-diabetic threshold. In other cases, the meanplasma glucose concentration is lowered from above the pre-diabeticthreshold to below the pre-diabetic threshold. In each case, the“threshold” refers to the plasma glucose concentrations discussed abovewith reference to the FPGT and the OGTT.

Methods of treating impaired glucose metabolism include both therapeuticand prophylactic treatment unless otherwise specified. For example, incases in which a patient is diagnosed with diabetes, it is desirable totreat the patient to achieve a therapeutically effective result, i.e., areduction in the individual's plasma glucose concentration to a levelbelow that classified as diabetic. Alternatively, in cases in which apatient is diagnosed with a glucose intolerance characterized aspre-diabetes, it is desirable to treat the patient via the methods ofthe present invention to prevent the individual's plasma glucose levelfrom rising to a level classified as diabetic, as well as reducing theindividual's plasma glucose level to or toward the normal range ofplasma glucose concentrations. In some cases, use of the methodsdescribed herein can be combined with other lifestyle changes, such asincreased exercise, to reduce plasma glucose levels from diabetic orpre-diabetic levels to within the normal range. Some methods of thepresent invention further comprise monitoring blood glucose levels ofthe patient (e.g., via a FPGT or an OGTT) to assess the patient'sresponse to the algal biomass treatment and the need for furtheradministration.

In some cases, an alternative treatment for diabetes (e.g., a non-algalpharmaceutical product), administered to a patient prior to treatmentaccording to the methods of the present invention, can be reduced oreliminated after treatment with Chlorella protothecoides microalgalbiomass. In some cases, the alternative treatment comprises apharmaceutical composition such as insulin, sulfonylureas (e.g.,glimepiride, glipizide, or tolazamide), biguanides (e.g., metformin),thiazolidinediones (e.g., pioglitazone or rosiglitazone), alphaglucosidase inhibitors (e.g., miglitol or acarbose), D-phenylalaninederivatives (e.g., nateglinide), Dipeptidyl peptidase-4 inhibitors(e.g., sitagliptin phosphate), or amylin or incretin mimetics (e.g.,pramlintide acetate or exenatide, respectively). In other cases, thealternative treatment is a non-algal dietary regime, dietaryrestrictions, or an exercise regime.

In some methods, a reduction in the mean plasma glucose concentration isaccompanied by a reduction in the percentage fat of total body weight inthe patient relative to the percentage prior to administration of thealgal biomass in accordance with the present invention. In some cases,the percentage fat of total body weight is reduced by at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least7%, at least 8%, at least 9%, or at least 10% relative to the percentagefat of total body weight prior to administration of the algal biomass.

In some methods in accordance with the present invention, Chlorellaprotothecoides microalgal biomass comprising at least 15% algal oil isadministered to a subject with a body mass index (BMI) of greater than24.9 or greater than 29.9 to reduce the percentage fat of total bodyweight and thereby reduce the individual's BMI relative to the BMI priorto administration of the algal biomass. The BMI is a single number thatevaluates an individual's weight status in relation to height, and ishighly correlated with body fat. Being overweight (characterized as aBMI of 25-29.9) or obese (characterized as a BMI of 30-39.9, or greaterthan 40 (extreme obesity)) is a significant factor in the developmentand/or prolongation of impaired glucose metabolism, including type 2diabetes. Ioannidis, I., Angiology 59(2 Suppl):39S-43S (2008), andKeller, U., Int J Vitam Nutr Res. 76(4):172-177 (2006).

A reduction in the percentage fat of total body weight in an individual,as discussed herein, will generally result in a reduction in theindividual's BMI as well. In some cases, an individual's BMI is reducedfrom a level indicating obesity or overweight to a level indicating anormal weight relative to height. Some methods of the present inventionfurther comprise monitoring a patient's percentage body fat and/or BMIto assess the patient's response to the algal biomass treatment and theneed for further administration.

In some embodiments, the reduced plasma glucose concentration and/or thereduction in percentage body fat is accompanied by an increase in therelative abundance of beneficial gut microflora, as discussed in greaterdetail below.

VIII. Methods of Increasing the Relative Abundance of Beneficial GutMicroflora

In another aspect, the present invention is directed to a method ofincreasing the relative abundance of beneficial gut microflora in asubject. In one embodiment, the method comprises first determining thatthe subject could benefit from treatment to increase the abundance ofbeneficial gut microflora and then administering to the subject aneffective regime of Chlorella protothecoides microalgal biomasscomprising at least 15% algal oil by dry weight. In such methods, themicroalgal biomass comprises a prebiotic, which supports an increase inthe relative abundance of particular microorganisms in thegastrointestinal tract of the individual subject.

A prebiotic, in contrast to a probiotic, which traditionally comprise adietary supplement containing the desired beneficial bacteria or yeastcells, is a composition that promotes the growth and propagation ofparticular species of microorganisms without actually ingesting themicrobes themselves. Lactobacillales are an order of gram-positivebacteria that comprise lactic acid bacteria, and are widely used in theproduction of fermented foods, including dairy products such as yogurtand cheese.

Approximately 100 species of microorganisms and 100 trillion or moreindividual microorganisms live in the human intestines and form theintestinal bacteria plexus. Intestinal bacteria, such as Lactobacillusbifidus and the like, have a strong relationship with the health ofhumans, while others have a detrimental effect on the body. Thedistribution of these flora varies with factors such as age, race,lifestyle, environment, diet, and the like. Intestinal flora inparticular are markedly affected by daily diet. Consequently, diet hasbeen promoted as of particular importance in the control of intestinalconditions. Commercial milk products, such as yogurt, containingLactobacillus bifidus for balanced intestinal function have been widelyused for many years. By means of these products, viable lactic acidbacteria are ingested in order to balance intestinal function (e.g.,preventing constipation).

As demonstrated in Example 10 below, administration of a diet comprisingalgal biomass derived from cultured Chlorella protothecoides results ina relative increase in the presence of gut microflora belonging to theclass Lactobacillales. The algal biomass functions as a prebiotic topromote the propagation of these beneficial microorganisms.

In some embodiments, methods in accordance with the present inventionfurther comprise monitoring the relative abundance of gut microflora ina subject to detect an increase in Lactobacillales or other beneficialgut microorganisms. Measurements can be made, as described in Example10, via use of terminal restriction fragment length polymorphism(T-RFLP) analyses to identify the relative abundance of variousmicroorganisms and assess the effect of the algal biomass regime.

IX. Dosage Forms

Administration of Chlorella protothecoides microalgal biomass inaccordance with the methods of the present invention can be performed byproviding to the patient or subject a microalgal biomass compositioncomprising an orally administrable dosage form. In some embodiments, theoral dosage form comprises a tablet or capsule, which may substitute foror supplement the subject's diet. In other embodiments, the oral dosageform comprises a food composition, which may be substituted for aportion of the subject's diet as a percentage by weight or by calories,or be used as a supplement.

A. Tablet and Capsule Formulations

In some cases, Chlorella protothecoides microalgal biomass can bemanufactured into nutritional or dietary supplements. For example,Chlorella protothecoides biomass or oil extracted from the biomass canbe encapsulated into digestible capsules in the manner similar to fibercapsules or fish oil. To prepare a tablet formulation for use in themethods of the present invention, Chlorella protothecoides microalgalbiomass, can be encapsulated into digestible capsules, or compressedinto a tablet by standard techniques familiar to those of skill in theart. In some cases, one or more excipients may be combined with themicroalgal biomass in the capsule or tablet. Such capsules or tabletscan be packaged in, e.g. a bottle or blister pack, and ingested on adaily basis or otherwise, as discussed in greater detail below withreference to administration schedules. In some cases, the tablet,capsule or other dosage formulation comprises a unit dose of biomass oralgal oil.

Manufacturing of capsule and/or tablet dosage forms is preferablyperformed under GMP conditions appropriate for drug products (ascodified at 21 CFR 210-211), nutritional supplements (as codified at 21CFR 111), or comparable regulations established by jurisdictions outsidethe United States.

B. Food Product Formulations

Preparation of food compositions for use in the methods of the presentinvention comprise combining Chlorella protothecoides microalgalbiomass, as described above, with at least one other edible ingredient,as described below, to form a food composition. In preferredembodiments, the preparatory methods are performed under GMP conditionsappropriate for drug products (as codified at 21 CFR 210-211), foodproducts (as codified at 21 CFR 110), or comparable regulationsestablished by jurisdictions outside the United States.

In various embodiments, the food composition prepared for use inaccordance with the methods of the invention comprises a baked good(e.g., cookies or a pie), a pasta product, a cake product, a breadproduct, an energy bar, a milk product, a juice product, or a smoothie.In various embodiments, the food composition weighs at least 10 g, atleast 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g,at least 70 g, at least 80 g, at least 90 g, at least 100 g, at least200 g, at least 300 g, at least 400 g, or at least 500 g or more. Insome embodiments, the food composition formed by the combination ofChlorella protothecoides microalgal biomass and at least one otheredible ingredient is an uncooked product. In other cases, the foodcomposition is a cooked product.

In some cases, the food composition formed by the combination ofChlorella protothecoides microalgal biomass and at least one otheredible ingredient comprises at least 1.5%, at least 5%, at least 10%, atleast 25%, or at least 50% w/w or v/v microalgal biomass. In someembodiments, food compositions formed as described herein comprise atleast 2%, at least 3%, at least 4%, at least 15%, at least 20%, at least30%, at least 35%, at least 40%, at least 45%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, or at least 95% w/w microalgal biomass. In somecases, the food composition comprises 5-50%, 10-40%, or 15-35% algalbiomass by weight or by volume.

In some cases, the food composition comprises predominantly intactChlorella protothecoides microalgal cells. In some cases, the algal oilis predominantly encapsulated in cells of the biomass. In other cases,the biomass comprises predominantly lysed cells.

Chlorella protothecoides microalgal biomass can be combined with one ormore other edible ingredients to make a food product. Alternatively, amanufacturer can sell Chlorella protothecoides microalgal biomass as aproduct, and a consumer can incorporate the algal biomass into a foodproduct, for example, by modification of a conventional recipe. Ineither case, the algal biomass can be used to replace all or part of theoil, fat, eggs, or the like used in many conventional food products.

Other edible ingredients with which algal biomass can be combined inaccordance with the present invention include, without limitation,grains, fruits, vegetables, proteins, meats, herbs, spices,carbohydrates, and fats. The other edible ingredients with which theChlorella protothecoides microalgal biomass is combined to form foodcompositions depend on the food product to be produced and the desiredtaste, texture and other properties of the food product. In some foodproducts, the microalgal biomass is combined with 2-20, 3-10, or 4-8other edible ingredients. In some food products, the microalgal biomassis combined with at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 other edibleingredients. The edible ingredients can be selected from all the majorfood groups, including without limitation, fruits, vegetables, legumes,meats, fish, grains (e.g., wheat, rice, oats, cornmeal, barley), herbs,spices, water, vegetable broth, juice, wine, and vinegar. In some foodcompositions, at least 2, 3, 4, or 5 food groups are represented as wellas the algal biomass or algal oil.

In some embodiments, the food product formulations comprise at least1.5%, at least 2%, at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least95% w/w or v/v Chlorella protothecoides microalgal biomass. Some foodcompositions comprise from 5-50%, 10-40%, or 15-35% microalgal biomassby weight or by volume. The remainder of a food composition inaccordance with the present invention comprises other conventionaledible ingredients, including those identified herein.

In cooked foods, the determination of percentages (i.e., weight orvolume) can be made before or after cooking. The percentage of Chlorellaprotothecoides microalgal biomass can increase during the cookingprocess because of loss of liquids. Because Chlorella protothecoidesmicroalgal biomass cells usually lyse in the course of the cookingprocess, it is difficult to measure the content of algal biomassdirectly in a cooked product. However, the content can be determinedindirectly from the mass or volume of biomass that went into the rawproduct as a percentage of the weight or volume of the finished product.

In uncooked foods, most Chlorella protothecoides microalgal cells in thebiomass remain intact. This has the advantage of protecting the algaloil from oxidation, which confers a long shelf-life. Depending on thenature of the food product, the protection conferred by the cells mayreduce or avoid the need for refrigeration, vacuum packaging or thelike. Retaining cells intact also prevents direct contact between theoil and the mouth of the subject administered the food productformulation. Alternatively, cells can be disrupted to release oil, whichcan provide the advantage of making the oil more available to perform astructural role in food preparation. Cells can be disrupted while in anaqueous suspension, which generally forms an emulsion, and also in dryform, which forms a free flowing powder of the cells are less than about60% oil by dry cell weight.

Food compositions for use in accordance with the methods of theinvention can include algal biomass with an oil content, an oilcomposition, and/or other constituents as described herein.

Chlorella protothecoides microalgal biomass for use in the methods ofthe present invention can also be formulated as a food ingredient, forcombination with at least one other edible ingredient by a subject priorto ingestion. Such microalgal biomass is preferably manufactured andpackaged under Good Manufacturing Practice (GMP) conditions for foodproducts as codified at 21 C.F.R. 110. In these instances, the Chlorellaprotothecoides microalgal biomass can be packaged in an airtightcontainer, such as a sealed bag. Optionally, the algal biomass can bepackaged under vacuum to enhance shelf life. Refrigeration of packagedalgal biomass is not required. The packaged Chlorella protothecoidesmicroalgal biomass can contain instructions for use including directionsfor how to combine the algal biomass with at least one other edibleingredient to prepare a food composition for administration inaccordance with the methods of the invention.

In some cases, Chlorella protothecoides microalgal biomass can bepackaged in a form combined with other dry ingredients (e.g., sugar,flour, dry fruits, flavorings). The mixture can then be converted into afood product by a consumer by addition of at least one other edibleingredient. In some cases, a liquid can be added to reconstitute a driedalgal biomass composition. Cooking can optionally be performed using amicrowave oven, convection oven, or conventional oven. Such mixtures canbe used for making cakes, breads, pancakes, waffles, drinks, sauces andthe like. Such mixtures have advantages of convenience for the consumeras well as long shelf life without refrigeration. Such mixtures aretypically packaged in a sealed container bearing instructions for addingliquid or other edible ingredients to convert the mixture into a foodproduct for use in the methods of the invention.

In some cases, Chlorella protothecoides biomass can also be packaged asready-to-mix material and presented in single use sachets or in bulk.Such ready-to-mix material can be combined with at least one other foodproduct that is intended for human consumption. As a non-limitingexample, Chlorella protothecoides biomass can be mixed with beveragessuch as water, juice, milk or other liquids. The Chlorellaprotothecoides biomass can also be mixed into food products such asyogurts.

X. Dose and Schedule of Administration

Effective doses of Chlorella protothecoides microalgal biomass can beadministered to a patient as a single daily dose, or can be administeredin a total daily dose divided into smaller doses administered two,three, four, or more times daily. In various embodiments, an effectivedose of algal biomass comprises from 1 gram to 100 grams of biomass perday. In some cases, an effective dose comprises from 1 gram to 250 gramsper day. In some cases, an effective dose of algal biomass comprisesfrom 1-5 grams of biomass per day. In some cases, an effective dosecomprises from 1-10 grams, from 5-20 grams, from 10-50 grams, from 20-75grams, or from 25-100 grams of biomass per day. In some embodiments, aneffective dose of algal biomass in accordance with the methods of thepresent invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 grams ofbiomass per day. In some cases, the effective dose comprises at leastthe amount identified in any one of the doses or dosage ranges discussedabove.

In some cases, an effective dose comprises from 1-2 grams of Chlorellaprotothecoides microalgal biomass per day. In other cases, an effectivedose comprises from 1.5-2.5, or from 2-3 grams of algal biomass per day.In still other cases, the effective dose is at least 1 g, at least 1.5g, at least 1.75 g, at least 2 g, at least 2.25 g, at least 2.5 g, atleast 2.75 g, or at least 3 g of algal biomass per day.

In some cases, the dose of Chlorella protothecoides microalgal biomasscomprises a defined percentage of the patient's diet by weight orcalories. In various embodiments, the percentage may range from 1-20%,from 2-15%, from 3-10%, from 2.5-5%, or the like. In some cases, thedaily dose of algal biomass comprises 1%, 1.5% 2%, 2.5%, 3%, 3.5%, 4%,4.5%, 5%, 5.5% 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% of the patient's daily diet byweight or by calories.

Administration of Chlorella protothecoides microalgal biomass to apatient can be performed according to a particular treatment schedule ordosing regimen. In some cases, Chlorella protothecoides microalgalbiomass is administered daily for at least one week, at least two weeks,at least three weeks, at least four weeks, at least one month, at leasttwo months, at least three months, at least six months, at least ninemonths, at least one year, at least two years, at least five years, orfor life.

In some cases, administration of a daily dose of Chlorellaprotothecoides microalgal biomass is proximate in time to intake of ameal. In these instances, administration of algal biomass with at leastone other edible ingredient in a food composition may be convenient forthe patient.

All references cited herein, including patents, patent applications, andpublications, are hereby incorporated by reference in their entireties,whether previously specifically incorporated or not. The publicationsmentioned herein are cited for the purpose of describing and disclosingreagents, methodologies and concepts that may be used in connection withthe present invention. Nothing herein is to be construed as an admissionthat these references are prior art in relation to the inventionsdescribed herein.

Although this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the use of Chlorella protothecoides microalgalbiomass following, in general, the principles of the invention andincluding such departures from the present disclosure as come withinknown or customary practice within the art to which the inventionpertains and as may be applied to the essential features hereinbeforeset forth.

XI. Examples

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1

Chlorella protothecoides strains from the University of Texas culturecollection were tested for growth on glycerol and glucose (UTEX 31, 249,250, 264). Each strain was inoculated from solid media into 25 ml liquidbase media (2 g/L yeast extract, 2.94 mM NaNO₃, 0.17 mM CaCl₂.2H₂O, 0.3mM MgSO₄.7H₂O, 0.4 mM K₂HPO₄, 1.28 mM KH₂PO₄, 0.43 mM NaCl) and grownshaking at 27° C. for 72 hours under a light intensity of 75 μEm⁻²s⁻¹.These cultures were used to inoculate each strain to a final density of1×10⁵ cells per ml into 24-well plates containing 2 ml of (a) base mediaonly; (b) base media plus 0.1% glucose; and (c) base media plus 0.5%reagent grade glycerol (EM Science, catalog #GX0185-6). Plates wereplaced in the dark and grown for 72 hours shaking at 27° C. Samples ofeach strain grown in the three conditions were diluted 1.9:1 indistilled H₂O and absorbance was read at 600 nm in a Molecular DevicesSpectraMax 340PC. All strains exhibited growth in the presence ofglucose and glycerol compared to only base media.

Example 2 Strains and Media

Chlorella protothecoides (STRAIN 250), (STRAIN 249), and (STRAIN 31)were obtained from the Culture Collection of Alga at the University ofTexas (Austin, Tex., USA). The stock cultures were maintained onmodified Proteose medium. Modified Proteose medium consisted (g/L) of0.25 g NaNO₃, 0.09 g K₂HPO₄, 0.175 g KH₂PO₄, 0.025 g CaCl₂.2H₂O), 0.075g MgSO₄.7H₂O and 2 g yeast extract per liter. Glycerol wastes frombiodiesel production (acidulated glycerol (AG) and non-acidulatedglycerol (NAG)) were obtained from Imperial Western Products (Selma,Calif., USA). “Pure” or “reagent grade” glycerol was from EM Science (adivision of Merck KGA), catalog # GX0185-6.

Experimental Design and Lipid Assay:

For each strain, 1 ml of following different media was prepared in24-well plates.

-   -   1. Proteose+1% pure glycerol+1% glucose    -   2. Proteose+1% pure glycerol+1% glucose (added after 72 hr)    -   3. Proteose+1% acidulated glycerol+1% glucose    -   4. Proteose+1% acidulated glycerol+1% glucose (added after 72        hr)    -   5. Proteose+1% non acidulated glycerol+1% glucose    -   6. Proteose+1% non acidulated glycerol+1% glucose (added after        72 hr)

Each strain was inoculated to different media to 5×10⁵ cells/mlconcentration. The cultures were kept in dark and agitated by orbitalshaker from Labnet (Berkshire, UK) at 430 rpm. After 72 hr of initialgrowth, 1% (w/v) glucose was added to #2, #4, and #6 media and culturedanother 24 hr. Dried cell-weight was measured in all samples. To measuredry cell weight, 1 ml of each culture was pelleted by centrifugation at5,000 rpm for 5 minutes in an Eppendorf 5415C centrifuge. After removingsupernatant, cell pellets were frozen at −80° C. and lyophilized in alab scale freeze dryer (Labconco, Mo., USA). After the cell pellets aredried, their weight was determined. Results are shown in FIG. 1.

Example 3 Strains and Media

Chlorella protothecoides (UTEX 31) was obtained from the CultureCollection of Algae at the University of Texas (Austin, Tex., USA). Thestock cultures were maintained on modified Proteose medium (see EXAMPLE2).

Experimental Design:

For each condition, 1 ml of following different media was prepared in24-well plates.

-   -   4. Proteose    -   5. Proteose+0.5% glucose    -   6. Proteose+0.5% xylose    -   7. Proteose+0.25% glucose+0.25% xylose

Chlorella protothecoides (UTEX 31) was inoculated to media containingdifferent sugars (glucose, or xylose) to 3×10⁵ cells/ml concentration.The cultures were kept in dark and agitated by orbital shaker fromLabnet (Berkshire, UK) at 430 rpm. After 72 hr of growth, cell growthwas measured by counting cell numbers of each culture. Results are shownin FIG. 2.

Example 4

Chlorella protothecoides strains (UTEX 250), (UTEX 249) and (UTEX 31))were obtained from the Culture Collection of Algae at the University ofTexas (Austin, Tex., USA). The stock cultures were maintained onmodified Proteose medium (see EXAMPLE 2). For each condition, 1 ml offollowing different media was prepared in 24-well plates.

-   -   1. Proteose    -   2. Proteose+1% glucose    -   3. Proteose+1% fructose

Each strain was inoculated to media containing different sugars(glucose, or fructose) to 1×10⁶ cells/ml concentration. The cultureswere kept in dark and agitated by orbital shaker from Labnet (Berkshire,UK) at 430 rpm. After 96 hr of growth, cell density was measured bycounting cell numbers of each culture. Results are shown in FIG. 3.

Example 5 Chlorella on Sucrose

Materials and Methods:

Chlorella protothecoides (UTEX 249) was inoculated into three 50 mlflasks of Proteose media with 1% sucrose (2.94 mM NaNO₃, 0.428 mMK₂HPO₄, 1.28 mM KH₂PO₄, 0.427 mM NaCl, 0.17 mM CaCl₂-2H₂O, 0.3 mMMgSO₄-7H₂O, proteose peptone 1 g/L) to a final cell density of 4×10⁵cells per ml. Invertase (Sigma #I4504) was added to two of the culturesat 0.01 U/ml and 0.05 U/ml. All three cultures were grown in the darkfor ˜60 hrs shaking at 150 rpm.

Results:

Final cell counts were performed on all three cultures after ˜60 hrs ofshaking in the dark. The control flask reached 4.4×10⁵ cells per mlwhile the 0.01 U/ml and 0.05 U/ml flasks reached cell densities of 1×10⁸and 3×10⁸ respectively. Each flask was checked for contamination at theend of the experiment by microscopic analysis and all were clean.

Example 6 Carbon Utilization Screens

Strains and Culture Conditions:

Seed cultures of the various strains of Chlorella protothecoidesidentified below were started as 1 ml liquid cultures in 24 well platesand were grown autotrophically for 48 hours in light, agitating at −350rpm. Plates were setup with 1.5% agarose-based solid Proteose media (seeEXAMPLE 2) containing 1% of glucose, glycerol, xylose, sucrose,fructose, arabinose, mannose, galactose, or acetate as the sole fixedcarbon source. For each strain, 5 μl of culture from the autotrophic 24well plate was spotted onto the solid media. Plates were incubated for 7days in the dark at 28° C. and examined for growth compared to a controlplate containing no additional fixed carbon. Growth was observed foreach of the species tested with each respective feedstock, as shown inTable 2 below. Growth of these strains on Proteose media in the dark inthe absence of additional fixed carbon either did not occur or wasextremely minimal

TABLE 2 Chlorella protothecoides grown on various fixed-carbonfeedstocks. Fixed Carbon Source Genus/Species Source/Destination GlucoseChlorella protothecoides UTEX 250 Glycerol Chlorella protothecoides CCAP211/8d Fructose Chlorella protothecoides UTEX 31 Fructose Chlorellaprotothecoides UTEX 250 Fructose Chlorella protothecoides CCAP 211/8dMannose Chlorella protothecoides UTEX 250 Galactose Chlorellaprotothecoides UTEX 25 Galactose Chlorella protothecoides UTEX 250Galactose Chlorella protothecoides UTEX 264 Acetate Chlorellaprotothecoides UTEX 31 Acetate Chlorella protothecoides UTEX 411 AcetateChlorella protothecoides CCAP 211/8d Acetate Chlorella protothecoidesUTEX 250

Example 7 Preparation of Biomass

Microalgal biomass is generated by culturing microalgae as described inany one of Examples 1-6 or through methods described herein such as Miaoand Wu, J. Biotechnology, 2004, 11:85-93. The microalgal biomass is thenharvested from the culture bioreactor, and washed with water to removeresidual salts and culture media.

GMP procedures are followed. Any person who, by medical examination orsupervisory observation, is shown to have, or appears to have, anillness, open lesion, including boils, sores, or infected wounds, or anyother abnormal source of microbial contamination by which there is areasonable possibility of food, food-contact surfaces, or food packagingmaterials becoming contaminated, is to be excluded from any operationswhich may be expected to result in such contamination until thecondition is corrected. Personnel are instructed to report such healthconditions to their supervisors. All persons working in direct contactwith the microbial biomass, biomass-contact surfaces, andbiomass-packaging materials conform to hygienic practices while on dutyto the extent necessary to protect against contamination of themicroalgal biomass. The methods for maintaining cleanliness include, butare not limited to: (1) wearing outer garments suitable to the operationin a manner that protects against the contamination of biomass,biomass-contact surfaces, or biomass packaging materials; (2)maintaining adequate personal cleaniness; (3) washing hands thoroughly(and sanitizing if necessary to protect against contamination withundesirable microorganisms) in an adequate hand-washing facility beforestarting work, after each absence from the work station, and at anyother time when the hands may have become soiled or contaminated; (4)removing all unsecured jewelry and other objects that might fall intobiomass, equipment, or containers, and removing hand jewelry that cannotbe adequately sanitized during periods in which biomass is manipulatedby hand. If such hand jewelry cannot be removed, it maybe covered bymaterial which can be maintained in an intact, clean, and sanitarycondition and which effectively protects against the contamination bythese objects of the biomass, biomass-contact surfaces, orbiomass-packaging materials; (5) maintaining gloves, if they are used inbiomass handling, in an intact, clean and sanitary condition. The glovesshould be of an impermeable material; (6) wearing, where appropriate, inan effective manner, hair nets, headbands, caps, beard covers, or othereffective hair restraints; (7) storing clothing or other personalbelongings in areas other than where biomass is exposed or whereequipment or utensils are washed; (8) confining the following to areasother than where biomass may be exposed or where equipment or utensilsare washed: eating foodstuffs, chewing gum, drinking beverages, or usingtobacco; (9) taking any other necessary precautions to protect againstcontamination of biomass, biomass-contact surfaces, or biomass-packagingmaterials with microorganisms or foreign substances including, but notlimited to, perspiration, hair, cosmetics, tobacco, chemicals, andmedicines applied to the skin. The microbial biomass can optionally besubjected to a cell disruption procedure to generate a lysate and/oroptionally dried to form a microalgal biomass composition.

The microalgal biomass can optionally be subjected to a cell disruptionprocedure to generate a lysate and/or optionally dried to form amicroalgal biomass composition. A variety of methods of cell disruptioncan be suitable including chemical, thermal, mechanical, mills,ultrasonication, homogenization or combinations thereof.

Example 8 Lipid Profile of Chlorella protothecoides

Growth:

Cultures of Chlorella protothecoides obtained from the CultureCollection of Algae at the University of Texas (Austin, Tex., USA), weremaintained and all experiments were carried out in Modified Proteasemedia (see EXAMPLE 2). For each strain, 10 ml cultures were setup in 50ml flasks as follows:

-   -   1. Protease growth media with no carbon addition;    -   2. Protease growth media with 1% glucose.

Chlorella protothecoides was grown in the two conditions describedabove, at an initial seeding density of 1.0×10⁶ cells/ml. The cultureswere kept in the dark and agitated at 250 rpm for 7 days. The cells wereharvested after a 7 day growth period, and assessed for growth in thedark relative to the control by measuring dried cell weight. Dry cellweights were determined as follows: One ml of culture was centrifugedand the resulting pellet was rinsed with water to remove any salt orresidual media; the final, rinsed pellet was frozen at −80 degree C.;and subjected to freeze drying overnight in a Freeze Dry System(Labconco, Mo., USA). Glycerolipid profile was determined by HPLCanalysis: Approximately 10 mg of dried biomass was mixed with 1 ml ofisopropanol saturated with KOH and incubated at 80° C. for 4 hours.Lipids from cell pellets were extracted and hydrolyzed using anisopropanol potassium hydroxide solution heated to 80° C. for fourhours. The extract samples were analyzed with an Agilent 1100 HPLC usingthe following method. The samples were derivatized with bromophenacylbromide (60 mg/ml) and loaded onto a Luna 5u C8(2) 100 A 150×2 mm column(Phenomenex). The samples were eluted from the column using a gradientof water to 100% Acetonitrile:tetrahydrofuran (95:5). Signals weredetected using DAD array detector at a wavelength of 254 nm. The resultsare expressed as a percentage of total lipids and are summarized belowin Table 3.

TABLE 3 Glycerolipid profiles of Chlorella protothecoides. Species C14:0 C 16:0 C 16:1 C 18:0 C 18:1 C 18:2 C 18:3 C 20:0 C 20:1 Chlorella0.57 10.30 0 3.77 70.52 14.24 1.45 0.27 0 protothecoides (UTEX 250)Chlorella 0.61 8.70 0.30 2.42 71.98 14.21 1.15 0.20 0.24 protothecoides(UTEX 25)

Example 9 Reduction of Plasma Glucose Levels and Body Weight Via AlgalBiomass Administration

Methods:

An F-Tank batch of Chlorella protothecoides (UTEX 250) (about 1,200gallons) was used to generate biomass. The batch (#ZA07126) was allowedto run for 100 hours, while controlling the glucose levels at 16 g/L,after which time the corn syrup feed was terminated. Residual glucoselevels dropped to <0 g/L two hours later. This resulted in a final ageof 102 hours. The final broth volume was 1,120 gallons. Both in-processcontamination checks and a thorough analysis of a final broth samplefailed to show any signs of contamination. The fermentation broth wascentrifuged and drum dried.

Forty five Syrian golden hamsters (Mesocricetus auratus; n=15 per group)were randomized to receive either control hypercholesterolemia inducingdiet alone, or the same diet with added Chlorella protothecoides biomassthat contained intact (undisrupted) cells. The Chlorella protothecoidesbiomass contained 22% lipid dry cell weight and was added to the diet asa dried powder. The compositions of the diets are shown in Table 4below. All animals were then fed the hypercholesterolemia inducing dietad libitum for 28 days with body weight and food consumption measuredevery three days. On day 25, energy expenditure, expressed as oxygenconsumption per gram body weight, was measured by indirect calorimetryusing a respiratory gas exchange system for rodents (MM-100 CWE, Inc.Pennsylvania, USA) Animals were then euthanized with an overdose ofsodium pentobarbital; body composition was determined immediately bydual emission x-ray absorptiometry (DEXA). The study protocol wasapproved by the University of Manitoba Animal Care Committee inaccordance to the Canadian Council on Animal Care Guidelines.

TABLE 4 Composition of hypercholesterolemia diets. Diet compositions(g/kg dry matter) Control 2.5% 5.0% Ingredients Diet C. ProtothecoidesC. Protothecoides Casein 200 200 200 Corn starch 260 235 210 Sucrose 330330 330 Lard/Sunflower Mix 50 50 50 Cellulose 105 105 105 DL-methionine5 5 5 Mineral mixture 35 35 35 Vitamin mixture 10 10 10 Cholinebitartrate 2 2 2 BTH 0.2 0.2 0.2 Cholesterol 2.5 2.5 2.5 Test Article 025 50 Total 1000 1000 1000

Blood was collected in heparinized tubes and separated into plasma andpacked red blood cells by centrifugation. Plasma glucose, triglycerides,total cholesterol and HDL cholesterol were measured using the VitrosChemistry System 350 (Ortho-Clinical Diagnostics, Johnson and Johnson,USA). Plasma insulin was measured by ELISA assay (Millipore, Mo., USA)using 10 μL of sample.

Results:

Table 5, below, shows the results of the physiological, biochemical, andpercentage body fat measurements obtained from the Syrian goldenhamsters administered the hyperglycemia-inducing diets with or withoutalgal biomass, as shown in Table 4.

TABLE 5 Physiological, biochemical and % body fat measurements. P vs.Control % Body Fat (% Total) Control 52.4 ± 1.3  2.5% Chlorellaprotothecoides 50.5 ± 1.4  NS 5.0% Chlorella protothecoides 48.3 ± 1.6 0.06 Oxygen Consumption (ml/g LBM) Control 1.56 ± 0.09 2.5% Chlorellaprotothecoides 2.20 ± 0.27 0.03 5.0% Chlorella protothecoides 2.12 ±0.26 0.05 Carbon Dioxide Production (ml/g LBM) Control 1.02 ± 0.06 2.5%Chlorella protothecoides 1.09 ± 0.09 NS 5.0% Chlorella protothecoides1.03 ± 0.08 NS Total Plasma Protein (g/L) Control 64.9 ± 1.5  2.5%Chlorella protothecoides 65.0 ± 1.1  NS 5.0% Chlorella protothecoides63.7 ± 1.1  NS Plasma Albumin (g/L) Control 32.0 ± 0.8  2.5% Chlorellaprotothecoides 32.3 ± 0.6  NS 5.0% Chlorella protothecoides 31.3 ± 0.7 NS

Consumption of C. protothecoides in a semi-purified hypercholesterolemicand hyperglycaemic-inducing diet decreased plasma glucose independent ofinsulin concentrations in the Syrian golden hamster model. Inparticular, the data show that incorporation of C. protothecoides into ahyperglycaemic dietary matrix improves insulin sensitivity, asillustrated by the 25% reduction in plasma glucose (see FIG. 4, P<0.05),without increasing circulating insulin concentrations (see FIG. 5,P>0.05) in the Syrian golden hamster. The data also show that consumingC. protothecoides in a food matrix exerts an effect on energyexpenditure through an increase in O₂ consumption without changing CO₂production (see Table 5). Body weight gain was not different betweengroups (see FIG. 6) but the consumption of C. protothecoides at 5%tended to reduce % total body fat (see Table 5, P=0.06), a finding whichis also linked to better glycaemic control. The consumption of C.protothecoides at 2.5% and 5% of total diet (w/w) did not result in anydifference between groups in the liver production of total plasmaprotein or albumin, the former usually modestly increased or decreasedin stress situations and the latter decreased in acute inflammationresponses (see Table 5). Consumption of this relatively high lipidbiomass did not affect blood lipids as compared to controls (see FIG. 4,P>0.05).

Chlorella protothecoides biomass, added to a food matrix, lowered bloodglucose levels by 25% in Syrian golden hamsters compared to controlsconsuming the same diet without added algal biomass. The percentage bodyfat tended to decrease with C. protothecoides consumption while O₂utilization tended to increase with C. protothecoides consumption,indicating possible changes in preferred energy substrate and metabolicrate.

Example 10 Increased Relative Abundance of Beneficial Gut Microflora ViaAlgal Biomass Consumption

Methods:

Forty five Syrian golden hamsters (Mesocricetus auratus; n=15 per group)were randomized to receive either control hypercholesterolemia inducingdiet alone, or the same diet with added Chlorella protothecoides biomassof the same lot used to formulate diets in Example 9, formulated asshown in Table 4 above. All animals were then fed thehypercholesterolemia inducing diet ad libitum for 28 days. On day 28,after the animals were euthanized by overdose with sodium pentobarbital,caecums were removed and immediately frozen in liquid nitrogen andstored at −80° C. until analyzed.

Terminal restriction fragment length polymorphism (T-RFLP) analysis wascarried out on the pooled contents of four caecums randomly selectedfrom the fifteen caecums stored for each group, and the resultscross-referenced with known DNA sequences form 16s rDNA clones. Theresults are summarized in Table 6 below.

T-RFLP uses natural variation in the 16s rDNA to classify thecomposition of a complex microbial microsystem in a hierarchal mannerfrom phylum→class→order. Genomic DNA extraction from mycobacterium hasbeen described in the art and can be suitable for extraction of DNA forT-RFLP. DNA extraction for the caecum samples was performed according tothe methods described in Kotlowski, et al., J. Med. Microbio. (2004) 53:927-933. Primers 27f (forward) 5′-GAAGAGTTTGATCATGGCTCAG-3′ (SEQ IDNO:1) and 342r (reverse) 5′-CTGCTGCCTCCCGTAG-3′ (SEQ ID NO:2) were usedin order to amplify part of the 16S rDNA gene. The forward primer werefluorescently labeled (WellRED D4dye, Sigma-Proligo, St. Louis, Mo.) toallow detection of the fragments by capillary electrophoresis. Thepolymerase chain reaction (PCR) was performed as follows: 94° C. for 1minute; 36 cycles at 94° C. for 1 minute; 55° C. for 1 minute; 72° C.for 2 minutes; and a final extension at 72° C. for 5 minutes.

Following the PCR amplification, the samples were subjected torestriction enzyme digestion in order to produce terminal restrictionfragments; the 27-342 region of 16S DNA was digested using HhaI (10 μlof PCR product, 10 unites of HhaI, 1× HhaI buffer and 20 μg of bovinserum). The mix was adjusted to a final volume of 20 μl with MilliQ(Millipore, Bedford, Mass.) water and the DNA was digested at 37° C. for3 hours. The precise length of terminal restriction fragments weredetermined by performing capillary electrophoresis with a CEQ 8800Genetic Analysis System (Beckman Coulter, Fullerton, Calif.). 2 μl offluorescently labeled fragments (from the restriction digests), 26 μl ofsample loading solution, and 0.5 μl of DNA size standard (400 bp) weremixed and separated using capillary electrophoresis. An electropherogramwith peaks of different sizes was obtained for each sample. Each peakrepresented an operational taxonomic unit (OTU) and was identified byits fragment size. However, the data produced from the specific DNAsequence does not always correspond to a known or culturable microbialspecies. Therefore, this particular analysis allows for qualitative orsemiquantitative description of microbial populations. Along with thechanges in phylogenetic diversity, the actual number of matches in theRibosomal Database Project II (accession number) also gives anindication of the diversity, as the total number of matches in thedatabase indicates a uniquely cloned terminal fragment. See RibosomalDatabase Project (RDP), Center for Microbial Ecology at Michigan StateUniversity; accessed on Jul. 23, 2008 athttp://rdp.cme.msu.edu/index.jsp. Changes greater than or equal to 2fold are generally considered biologically significant Similaranalytical techniques have been described in Sepehri et al., InflammBowel Dis (2007), 13(6): 675-683 and are hereby incorporated byreference for all purposes.

Results:

Table 6 shows the relative changes in caecal microflora in response toingestion of C. protothecoides biomass, as described above. Relativechanges were calculated by setting the control group hamster data as the100% and measuring the relative fold changes for the 2.5% and 5.0% C.protothecoides groups. The greater than 5 fold change in the classLactobacillales compared to control hamsters indicates a positive andhealthy change in gut microflora. Generally regarded as healthy,bacteria from the class Lactobacillales are normally found in fermenteddairy products. More recently, the food industry has started to includevarious species (single or in combination) from this particular order infood preparation under the functional food category “probiotics”.Therefore, a “prebiotic” capable of inducing the greater than 5 foldchanges in favour of this particular species, in vivo, is significant,especially given the absence of these bacteria in the diet.

Other noteworthy changes include a different microbial pattern for classGammaproteobacteria. Specifically, control animals had higher relativenumbers from the order Pasteurellales, which contains normal gutcommensal bacterial species to pathogenic species, such as Haemophilusinfluenza, which is known to cause infection like pneumonia andbacterial meningitis. The consumption of 2.5% and 5.0% C. protothecoidesalso resulted in a greater than 2 fold increase in classEnterobacteriales, which includes Escherichia coli. Members of thisparticular class are commonly found in the intestinal microflora. Otherchanges included differences in the class Clostridia. Finally, a 7 foldincrease in the class Lentisphaerae was noted in the 2.5% C.protothecoides hamsters, while the class was not detected in the controlor 5% C. protothecoides.

These data support the conclusion that algal biomass comprisingsignificant quantities of algal oil (e.g., at least 15%) acts as aprebiotic for Lactobacillales species, and that consumption of suchalgal biomass increases the relative size of this population in the gut.

TABLE 6 Relative changes in caecal micorflora. 2.5% 5.0% C. C. BacterialClassification Control protothecoides protothecoides Total AccessionNumbers* 71 (1) 149 (2.1) 127 (1.8) phylum Lentisphaerae none 7 nonedetected detected class Lentisphaerae none 7 none detected detectedphylum Firmicutes 1 1 1 class Bacilli 1 5.3 5.6 order Lactobacillales 15.3 5.6 class Clostridia 1 0.94 0.92 order Clostridiales 1 0.82 0.89unclassified Clostridia 1 4.8 1.8 phylum Proteobacteria 1 0.96 1.1 classGammaproteobacteria 1 2.4 2.8 order Pasteurellales 1 0.50 0.57 orderEnterobacteriales none 2.7 3.1 detected unclassified Proteobacteria none7 8 detected *Total accession numbers refers to the total number ofunique matches in the RDP II database. This value is also reflective ofthe change in diversity for the microbial population of the caecum.

Example 11 Formulation of Chlorella protothecoides Biomass Suitable forHuman Consumption

Chlorella protothecoides biomass is prepared under conditions describedabove in Example 7. The biomass can be formulated in an encapsulatedform or as a tablet that is suitable for human consumption. Biomassformulated in an encapsulated form or as a tablet ideally should bemanufactured under GMP conditions appropriate for nutritionalsupplements as codified at 21 C.F.R. 111, or comparable regulationsestablished by foreign jurisdictions. The dried Chlorella protothecoidesbiomass can also be mixed in with foods such as yogurt, breakfastcereal, salads, salad dressings, etc. or in beverages such as shakes,juice drinks or smoothies. The biomass should be taken at a level whereincreased probiotic levels can be observed.

Example 12 Genotyping of Chlorella protothecoides

Genomic DNA was isolated from the following Chlorella protothecoidesstrains: UTEX 25, UTEX 249, UTEX 250, UTEX 256, UTEX 264, UTEX 411, CCAP211/17 and CCAP 211/8d. Cells (approximately 200 mg each) werecentrifuged from liquid cultures for 5 minutes at 14,000×g. Cells werethen resuspended in sterile distilled water, centrifuged for 5 minutesat 14,000×g and the supernatant discarded. A single glass bead ˜2 mm indiameter was added to the biomass and tubes were placed at −80° C. forat least 15 minutes. Samples were removed and 150 μl of grinding buffer(1% Sarkosyl, 0.25M Sucrose, 50 mM NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH8.0, 0.5 μg/μl RNase A) was added. Pellets were resuspended by vortexingbriefly, followed by the addition of 40 μl of 5M NaCl. Samples werevortexed briefly, followed by the addition of 66 μl of 5% CTAB (Cetyltrimethylammonium bromide) and a final brief vortex. Samples were nextincubated at 65° C. for 10 minutes after which they were centrifuged at14,000×g for 10 minutes. The supernatants were transferred to freshtubes and extracted once with 300 μl Phenol: Chloroform:Isoamyl alcohol12:12, followed by centrifugation for 5 minutes at 14,000×g. Theresulting aqueous phase was transferred to a fresh tube containing 0.7vol of isopropanol (˜190 μl), mixed by inversion and incubated at roomtemperature for 30 minutes or overnight at 4° C. DNA was recovered viacentrifugation at 14,000×g for 10 minutes. The resulting pellet was thenwashed twice with 70% ethanol, followed by a final wash with 100%ethanol. Pellets were air dried for 20-30 minutes at room temperaturefollowed by resuspension in 50 μl of 10 mM TrisCl, 1 mM EDTA (pH 8.0).

Five μl of total DNA, prepared as described above, from each Chlorellaprotothecoides strain, was diluted 1:50 in 10 mM Tris, pH 8.0. PCRreactions, final volume 20 μl, were set up as follows: Ten μl of 2×iProof HF master mix (Bio-Rad) was added to 0.4 μl primer SZ02613(5′-TGTTGAAGAATGAGCCGGCGAC-3′ (SEQ ID NO:3) at 10 mM stockconcentration). This primer sequence runs from position 567-588 inGenBank accession no. L43357 and is highly conserved in higher plant andalgal plastid genomes. This was followed by the addition of 0.4 μlprimer SZ02615 (5′-CAGTGAGCTATTACGCACTC-3′ (SEQ ID NO:4) at 10 mM stockconcentration). This primer sequence is complementary to position1112-1093 in GenBank accession no. L43357 and is highly conserved inhigher plants and algal plastid genomes. Next, 5 μl of diluted total DNAand 3.2 μl dH₂O were added. PCR reactions were run as follows: 98° C.,45″; 98° C., 8″; 53° C., 12″; 72° C., 20″ for 35 cycles followed by 72°C. for 1 minute and holding at 25° C. For purification of PCR products,20 μl of 10 mM Tris, pH 8.0, was added to each reaction, followed byextraction with 40 μl of Phenol: Chloroform:Isoamyl alcohol (12:12:1),vortexing and centrifuging at 14,000×g for 5 minutes. PCR reactions wereapplied to S-400 columns (GE Healthcare) and centrifuged for 2 minutesat 3,000×g. Purified PCR products were subsequently TOPO coloned intoPCR8/GW/TOPO and positive clones selected for on LB/Spec plates.Purified plasmid DNA was sequenced in both directions using M13 forwardand reverse primers. Sequence alignments and results are summarized inCladograms in FIGS. 7a-7c . Sequences from all eight strains ofChlorella protothecoides are listed as SEQ ID NO:5 and SEQ ID NO:6 inthe attached Sequence Listing.

What is claimed is:
 1. A method of increasing the relative abundance ofbeneficial gut microflora in a subject, comprising determining thesubject would benefit from a greater abundance of beneficial gutmicroflora and then administering to the subject an effective regime ofChlorella protothecoides microalgal biomass comprising at least 15%algal oil by dry weight, whereby the relative abundance of beneficialgut microflora is increased as compared to the relative abundance beforeadministering the regime.
 2. The method of claim 1, wherein the algalbiomass contains 25-75%, of algal oil by dry weight.
 3. The method ofclaim 1, wherein at least 50% by weight of the algal oil ismonounsaturated oil.
 4. The method of claim 1, wherein less than 5% byweight of the algal oil is docosahexanoic acid (DHA).
 5. The method ofclaim 1, wherein the algal oil is predominantly encapsulated in cells ofthe biomass.
 6. The method of claim 1, wherein the biomass isadministered as a homogenate.
 7. The method of claim 1, wherein theregime comprises administering the algal biomass at a dose of 1-20% offood by weight or calories.
 8. The method of claim 1, wherein the algalbiomass is administered daily for at least a week.
 9. The method ofclaim 1, wherein the algal biomass is administered with at least oneother edible ingredient as a food composition.
 10. The method of claim1, wherein the regime additionally lowers the percentage fat of totalbody weight in the subject relative to the percentage before theadministering step.
 11. The method of claim 1, wherein the algal biomassis derived from Chlorella protothecoides grown heterotrophically. 12.The method of claim 11, wherein the Chlorella protothecoides is grown ina culture medium including a feedstock comprising at least one carbonsubstrate selected from the group consisting of a depolymerizedcellulosic material, a 5-carbon sugar, and a 6-carbon sugar.
 13. Themethod of claim 1, wherein the algal biomass is administered in the formof a tablet or capsule.
 14. The method of claim 1, wherein the algalbiomass is administered in the form of a food product.